Discovery of N-Aryloxypropylbenzylamines as Voltage-Gated Sodium Channel NaV1.2-Subtype-Selective Inhibitors

Article abstract of DOI:10.1002/cmdc.201800781

We previously reported that a lipophilic N-(4′-hydroxy-3′,5′-di-tert-butylbenzyl) derivative (1) of the voltage-gated sodium channel blocker mexiletine, was a more potent sodium channel blocker in vitro and in vivo. We demonstrate that replacing the chiral methylethylene linker between the amine and di-tert-butylphenol with an achiral 1,3-propylene linker (to give (2)) maintains potency in vitro. We synthesized 25 analogues bearing the 1,3-propylene linker and found that minor structural changes resulted in pronounced changes in state dependence of blocking human Na_V1.2 and 1.6 channels by high-throughput patch-clamp analysis. Compared to mexiletine, compounds 1 and 2 are highly selective Na_V1.2 inhibitors and >500 times less potent in inhibiting Na_V1.6 channels. On the other hand, a derivative (compound 4) bearing 2,6-dimethoxy groups in place of the 2,6-dimethyl groups found in mexiletine was found to be the most potent inhibitor, but is nonselective against both channels in the tonic, frequency-dependent and inactivated states. In a kindled mouse model of refractory epilepsy, compound 2 inhibited seizures induced by 6 Hz 44 mA electrical stimulation with an IC₅₀ value of 49.9±1.6 mg kg^?1. As established sodium channel blockers do not suppress seizures in this mouse model, this indicates that 2 could be a promising candidate for treating pharmaco-resistant epilepsy.

Full text of DOI:10.1002/cmdc.201800781

Accepted Article
Title: Discovery of N-Aryloxypropylbenzylamines as Voltage-gated
Sodium Channel NaV1.2 subtype Selective Inhibitors
Authors: Phillip L. van der Peet, Saman Sandanayake, Bevyn Jarrott,
and Spencer John Williams
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201800781
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800781
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Discovery of N-Aryloxypropylbenzylamines as Voltage-gated
Sodium Channel NaV1.2 subtype Selective Inhibitors
Phillip L. van der Peet,[a] Saman Sandanayake,[a] Bevyn Jarrott,*[b] Spencer J. Williams*[a]
[a]
Dr P. van der Peet, Dr S. Sandanayake, Prof. S.J.
Williams 0000-0001-6341-4364
School of Chemistry and Bio21 Institute of Molecular Science and Biotechnology
University of Melbourne
Parkville, Vic. 3010, Australia
E-mail: sjwill@unimelb.edu.au
[b]
Prof. B. Jarrott
Florey Institute of Neuroscience & Mental Health
Parkville, Vic. 3010, Australia
E-mail: bevyn.jarrott@florey.edu.au
Supporting information for this article is given via a link at the end of the document.
Abstract: We previously reported that a lipophilic N-(4’-hydroxy-3’,5’-
di-tert-butylbenzyl) derivative (1) of the voltage-gated sodium channel
blocker mexiletine, was a more potent sodium channel blocker in vitro
and in vivo. We demonstrate that replacing the chiral methylethylene
linker between the amine and di-tert-butyl-phenol with an achiral 1,3-
propylene linker (to give (2)) maintains potency in vitro. We
synthesised 25 analogues bearing the 1,3-propylene linker and found
that minor structural changes resulted in pronounced changes in state
dependence of blocking human NaV 1.2 and 1.6 channels by high
throughput patch-clamp analysis. Compared to mexiletine,
compounds 1 and 2 are highly selective NaV1.2 inhibitors and >500
times less potent in inhibiting NaV1.6 channels. On the other hand, a
derivative (4) bearing 2,6-dimethoxy groups in place of the 2,6-
dimethyl groups found in mexiletine was the most potent inhibitor but
is non-selective against both channels in the tonic, frequency-
dependent and inactivated states. In a kindled mouse model of
refractory epilepsy compound 2 inhibited seizures induced by 6 Hz 44
mA electrical stimulation with an IC50 value of 49.9 ± 1.6 mg/kg. As
established sodium channel blockers do not suppress seizures in this
mouse model, this indicates that 2 could be a promising candidate for
treating pharmaco-resistant epilepsy.
that can lead to disorders such as epilepsy, neuropathic pain,
arrhythmias, migraine, hyperthermia and other syndromes.[3]
Blockade of Nav by low molecular weight organic compounds has
been used to manage but not cure these clinical conditions by
inhibiting pathological firing patterns of neurons.
Voltage-gated sodium channels can exist in three main
states: deactivated (closed), activated (open), or inactivated
(closed).[4-5] In normal function, prior to an action potential, the
axonal membrane is at its normal resting potential with sodium
channels in the deactivated state. In response to membrane
depolarization, the channel is activated, allowing sodium ions to
flow into the neuron and causing further depolarization giving rise
to action potential. At the peak of the action potential, the channel
is inactivated and closes, stopping conduction of sodium ions and
allowing repolarization of the axon. As the membrane potential
falls, the channel returns to the deactivated state, ready to
participate in another action potential.
Axons express two subtypes of sodium channels: NaV1.2
and NaV1.6 and these have different localizations. NaV1.6
channels cluster in the nodes of Ranvier, where they trigger
saltatory conduction and also in the distal end of the axon initial
segment, where they promote action potential initiation along the
axon.[6] Conversely, NaV1.2 channels are densely localized in the
proximal axon initial segment, where they promote back
propagation to the soma and dendrites,[7] with preliminary
medicinal chemistry leading to the development of NaV1.2-
selective drugs.[8] Additionally NaV1.6 channels are present in
microglia adjacent to axons and can trigger release of
inflammatory cytokines in multiple sclerosis. Thus drugs blocking
these subtypes could be neuroprotective against axonal damage.
For example, CFM6058 is a selective inhibitor of NaV1.6 and
provides potent neuroprotective activity in hippocampal slices
(Figure 1).[9]
Introduction
Ion channels are heteromeric proteins that form gated, water-filled
pores that span lipid bilayer plasma membranes and subcellular
organelles and allow rapid movement of charged ions into and out
of excitable cells in response to chemical stimuli, temperature
changes or mechanical forces.[1] They are a large family of
proteins with more than 300 genes encoding the subunits that
confer different biochemical, biophysical and pharmacological
properties, such as ion passage selectivity and inhibitor
selectivity.[2] The proteins are dynamic and can adopt different
conformations depending on membrane potential enabling the
control of ion movement across the membrane. In mammals, one
important class of ion channels are the voltage-gated sodium
channels (NaV), which are responsible for the initiation of electrical
signalling in nerves and muscles. Nine isoforms of NaV have been
identified in humans; these are designated NaV 1.1 to NaV 1.9 and
they have a high degree of sequence homology.[3] Furthermore,
over 1,000 mutations have been identified in human NaV channels
1
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more potent than mexiletine in displacing the radioligand [3H]-
batrachotoxinin in binding to Na+ channels in depolarised rat brain
membranes, and in vivo, was more effective than mexiletine as a
drug candidate in reducing pain behaviors in three different rat
models of neuropathic pain (formalin paw model, ligated spinal
nerve model, and contusive spinal cord injury model).[18] HFI-1
shares the 3,5-tert-butyl-4-hydroxyphenylmethyl substituent with
AM-36, a neuroprotective agent that has combined antioxidant
and Na+ channel blocking activity.[19]
In view of the potency of 1 as a NaV ligand and a drug
candidate we synthesized analogues of 1 designed to simplify the
branched (and chiral) 2-aminopropyloxy linker within the molecule
and determined the subsequent structure-activity relationships.
We assessed the potency of these analogues for displacing [3H]-
batrachotoxinin from Na+ channels in rat brain membranes, and
then assessed the ability for these molecules to influence the
electrophysiological properties of human NaV1.2 and NaV1.6
sodium channels expressed in Chinese hamster ovary cells. The
most promising compound was evaluated in a mouse model of
pharmacoresistant epilepsy.
Figure 1. Selected sodium channel blockers.
Results and Discussion
Mexiletine was originally developed as
a class 1B
antiarrhythmic drug for the control of ventricular arrhythmias,[10-11]
and is a frontline drug for the treatment of myotonic syndromes
and possesses activity on the product of the SCN4A gene,
NaV1.4.[12] Early infantile epileptic encephalography has been
found to be due to a mutation in the SCN2A gene expressing
NaV1.2 that causes a gain of function in the brain leading to tonic-
clonic partial seizures and oral mexiletine has been found to
reverse the epileptogenic dysfunction in these infants.[13] Also, it
has been shown to relieve allodynia and hyperalgesia in rodent
models of neuropathic pain, and affords relief in patients with
neuropathic pain at relatively high doses (150-900 mg/d).[14]
However, patient acceptance is poor with gastrointestinal nausea
reported by 40% of patients.
Mexiletine represents an interesting lead compound for
development of new NaV blockers as it has high potency, yet has
a low molecular weight, allowing for room to modify its structure
without exceeding MW guidelines for drug-like compounds.
Previous studies have reported variations in the aryloxy group and
the linker of mexiletine. Carocci and co-workers showed that
meta-hydroxymexiletine was equipotent on skeletal NaV channels,
and possessed superior potency on cardiac NaV channels, and
Desaphy and co-workers showed that hydroxylation of the aryloxy
ring led to variations in potency of the resultant drugs that
correlated with lipophilicity.[15] Conte-Camerino and co-workers
showed that variations in the methylethylene group, by
replacement of the methyl group with lipophilic substituents, led
to derivatives more potent than mexiletine in use- and voltage-
dependent block in frog muscle fibers.[16] Relevant to this work,
the 2-methylpropyleneamine derivative of mexiletine exhibited
similar potency to mexiletine in use-dependent block, while
introduction of a 4-chloro group in Me2-Cl provided enhanced
tonic and use-dependent block.[17]
Chemistry: Design and Synthesis
Compound 1 was designed to increase the lipophilicity of
mexiletine to allow analogues to gain access to sodium channels
within the central nervous system.[18] This was achieved by
appending the antioxidant di-tert-butylphenol group, to allow
inclusion of an antioxidant fragment that has previously been
shown with AM-36 to reduce oxidative stresses within the CNS.[20-
21]
In developing a series of analogues of 1 we initially aimed to
simplify the structure by replacing the branched 2-aminopropyloxy
linker, which contains a stereogenic centre, with a linear 1,3-
propylene linker, thereby avoiding issues with chirality. We initially
prepared compound 2 by a three-step approach involving
alkylation of 2,6-dimethylphenol with 3-bromopropylphthalimide,
followed by removal of the phthalimide group by hydrazinolysis,
and finally reductive amination with 3,5-di-tert-butylbenzaldehyde
(Figure 2a). When assessed for binding to rat brain by
displacement of the radioligand [3H]-batrachotoxinin, a similar IC50
value was obtained relative to 1 (Table 1, vide infra). This
promising result encouraged the preparation of additional
derivatives maintaining the structure of the 3-aminopropyloxy
linker and exploring variation in the nature of the left hand side
aryloxy group, and the right hand side phenolic group.
Mexiletine has a low partition coefficient (logD = 0.39 at pH 7.4),
which likely restricts its passage into the central nervous system.
An analogue of mexiletine, HFI-1 (1), was developed with greater
lipophilicity (logD = 5.53 at pH 7.4).[18] In vitro, HFI-1 was 82-fold
2
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tBu
OH
H
N
Ar1O
tBu
Compound
Mexiletine
1 (HFI-1)
Ar1O
IC50
(
µ
M)
-
-
11
0.13
0.18
2
3
0.10
0.11
Figure 2. Synthesis of aryloxypropylbenzylamine derivatives.
Analogues that varied in the structure of the left-hand aryloxy
group (Ar1) were readily prepared from various commercially
available phenols using the route in Figure 2a, and were chosen
to explore variation in the size and nature of the 2,6-substituents,
as well as the effect of introducing additional substituents (methyl,
isopropyl, phenyl, thiophenyl) or nitrogen into the ring.
Alternatively, the 2,6-dimethylphenyl ring was replaced by a
pyrazole heterocycle. We also investigated analogues that varied
the nature of the right-hand group (Ar2) whilst maintaining the 2,6-
dimethylphenyl group. These compounds were prepared from the
aryloxypropylamine by reductive amination (Figure 2b).
4
5
0.09
0.38
6
Binding to the batrachotoxinin site in rat brain
membranes
Voltage-sensitive sodium channels have a range of receptor sites
for neurotoxins. Batrachotoxinin interacts with site 2, causing
persistent activation of sodium channels, and is competitive with
a range of effective sodium channel blocking drugs.[18] IC50 values
for inhibition of binding of [3H]-batrachotoxinin is shown for
mexiletine and 14 analogues in Table 1. As we have previously
reported, mexiletine competes with [3H]-batrachotoxinin for
binding to washed rat brain membranes with an apparent IC50
value of 11 µM, and appending the di-tert-butylphenoxy group to
afford 1 results in an 82-fold enhancement of activity.9 Conversion
of the 2-methylethylene linker to the achiral propylene linker in 2
provided a small attenuation in activity relative to 1, but was still
61-fold more potent than mexiletine. A range of variations to the
2,6-dimethylphenyl group were explored, including variation of the
2,6-dimethyl groups (3-5), conversion to a pyridine or pyrimidine
heterocycle (6, 8, 13, 14), and introduction of substituents at the
3 and 4 positions (9,10,11). In general, most variations led to
either retention or reduction in activity. Notably, the 2,3,6-
trimethylphenyl group (compound 12) provided an approximately
2- and 3-fold enhancement in activity relative to compounds 1 and
2, respectively.
7
8
0.24
0.87
9
0.15
0.28
10
11
12
0.12
0.056
Table 1. IC50 values for Ar1 variants binding to sodium channel site 2 in rat brain
membranes.[a]
3
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13
14
0.84
0.27
[a] IC50 values for binding to sodium channel site 2 in rat brain membranes
determined in competition with [3H]-batrachotoxinin.
Table 2 shows results for binding to sodium channel site 2
in rat brain membranes in competition with [3H]-batrachotoxinin
upon initial variation of the di-tert-butyl-4-hydroxyphenyl group.
Removal of the tert-butyl groups to provide 15 gave a ~10-fold
loss of activity relative to 1, and replacement with chloro groups
16 led to loss of activity. Interestingly, the phenyl analogue 17 was
only ~2-fold less potent than 2; in this case conversion of the di-
tert-butylphenoxy group of 2 to the phenyl of 17 results in a
relatively large change in calculated LogD (pH 7.4): 2, 3.7; 17, 2.2.
The acetamido derivative 18 was inactive.
4
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Table 2. IC50 values for Ar2 variants binding to sodium channel site 2 in rat brain
membranes.[a]
Compound
Mexiletine
1 (HFI-1)
Ar2
IC50
(
µ
M)
-
-
11
0.13
1.19
15
16
Figure 3. Patch clamp voltage protocols. a) Protocol 1 used for assessing state
dependence of NaV1.2 and 1.6 channels. The test-pulse potential was 0 mV for
NaV1.2; 10 mV for NaV1.6. b) Protocol 2 allows assessment of contribution of
binding to slow inactivated channels.
>0.9
0.404
>0.9
17
18
LHS structure-activity relationships: Inclusion of a phenyl or 2-
thiophenyl group (compounds 11, 19) at the para position led to a
loss of activity against NaV1.2; compound 20, which possesses a
3-thiophenyl group displayed some inhibition for the tonic and
inactivated states. On the other hand, most other compounds
displayed a marked enhancement in activity in binding to NaV1.2
versus mexiletine for the tonic and 10 Hz states, and similar
potencies for the inactivated states. Unlike mexiletine, which is a
highly selective inhibitor of the inactivated state, most compounds
displayed similar potencies across the three states. Interestingly,
the trimethylpyrazole 22 is selective for the 10 Hz and inactivated
states. For NaV1.6, important differences are evident.
Compounds 2, 5, 10, 11, 19, 20, and 23 did not significantly inhibit
NaV1.6. The structural factors responsible for lack of binding are
complex, but it is clear that para-substituents are not tolerated.
Compound 1 possessed similar activity towards NaV1.6 as
mexiletine, the remaining active compounds were much more
potent than either compound, and mostly showed little selectivity
across the three states. Notable exceptions include compounds
13 (>2-3-fold selectivity for 10 Hz and inactivated states) and 22
(9- and 16-fold selectivity for the 10 Hz and inactivated states).
While mexiletine is 2-fold selective for NaV1.2 versus NaV1.6,
many of the compounds display greatly altered selectivity.
Notably, the data for compounds 1 and 2 reveal that changing the
2-methylethylene linker to the 1,3-propylene linker results in
compounds with similar potency for NaV1.2, but an enhancement
in NaV1.2:1.6 selectivity from approx. 1:80, to approx. 1:250 or
better across all three states. For the remainder of the active
compounds, selectivity for the two channels was more modest
and usually in favour of NaV1.2. However, compounds 8, 13, 21,
and 22 exhibited selectivites either close to unity or slightly in
favour of NaV1.6. The most potent compound of the series was
compound 4, which displayed sub-micromolar inhibition of all
states of the two channels.
[a] IC50 values for binding to sodium channel site 2 in rat brain membranes
determined in competition with [3H]-batrachotoxinin.
Electrophysiological assessment of binding to NaV
1.2 and 1.6
To gain greater insight into binding of the full suite of compounds
1-26 at specific channels important in the brain, we studied the
state dependence of blocking of human NaV1.2 and 1.6 channels
using a high throughput patch-clamp system. Initial studies were
performed using an Ionworks Quattro instrument. The voltage
stimulus pattern is shown in Figure 3a. Test pulses applied at the
indicated times allowed determination of tonic block (binding to
resting closed channels); frequency-dependent block at
a
stimulation frequency of 10 Hz, and slow inactivated state block.
All compounds were screened using protocol 1 as the primary
screen. The results are shown in Tables 3 and 4. Initially,
screening for compounds 1-8 were performed at a concentration
range of 0.1–1000 µM; all other compounds were subsequently
screened at a lower concentration range of 0.1–30 µM.
5
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Table 3. Variation of Ar1 on NaV1.2 and NaV1.6 channel affinities (Protocol 1).
Slow
inactivated
3.9
>10
-
Block type
NaV1.2
IC50
NaV1.6
IC50
NaV1.2
Ar1
selectivity
[a]
(
µ
M)
( M)
µ
11
12
13
14
19
20
21
22
23
Tonic
10 Hz
>10
>10
>10
>10
-
-
Mexiletine
Tonic
10 Hz
122
256
101
2.1
2.0
-
Slow
inactivated
51.8
>10
>10
-
Slow
inactivated
Tonic
10 Hz
3.1
3.5
>30
>30
>10
-
8.2
23.5
2.9
Tonic
10 Hz
1
3.6
3.9
316
324
88
83
-
Slow
inactivated
3.8
>30
-
Tonic
10 Hz
>10
>10
5.1
-
Slow
inactivated
3.9
311
80
≈10.6
0.5
2
3
Tonic
10 Hz
4.1
3.3
>1000
>1000
>250
>300
Slow
inactivated
5.0
3.5
0.7
Tonic
10 Hz
3.9
2.4
14.3
12.8
3.7
5.3
Slow
inactivated
2.4
>1000
>400
Tonic
10 Hz
2.0
2.3
6.0
6.6
3
Slow
inactivated
1.7
11.2
6.6
2.9
Tonic
10 Hz
>10
>10
>10
>10
-
-
Slow
inactivated
2.1
5.3
2.5
4
Tonic
10 Hz
0.3
0.4
0.7
0.8
2.3
2.0
Slow
inactivated
>10
>10
-
Tonic
10 Hz
8.3
9.1
>10
>10
-
-
Slow
inactivated
0.5
0.6
1.2
5
Tonic
10 Hz
6.3
6.8
>10
>10
>1
>1
Slow
inactivated
>10
>10
-
Tonic
10 Hz
9.0
2.9
4.8
2.0
0.5
0.7
Slow
inactivated
7.4
>10
>1
7
Tonic
10 Hz
2.8
2.5
4.2
3.5
1.5
1.4
Slow
inactivated
2.3
1.3
0.6
Tonic
10 Hz
>30
4.7
29.0
3.2
-
Slow
inactivated
2.0
2.6
1.3
0.7
8
Tonic
10 Hz
>10
9.5
>10
7.5
-
Slow
inactivated
3.2
1.8
0.6
0.8
Tonic
10 Hz
5.9
7.0
>10
>10
-
-
Slow
inactivated
5.7
5.9
1.0
9
Tonic
10 Hz
5.6
4.2
7.3
6.2
1.3
1.5
Slow
inactivated
7.9
>10
-
Slow
inactivated
[a] In some cases the selectivity could not be calculated; an estimate is provided
if >10.
2.8
6.3
2.3
10
Tonic
10 Hz
3.1
3.5
>10
>10
-
-
RHS structure-activity relationships: Compound 15, lacking
the t-butyl groups present in compound 2, maintained activity at
NaV1.2 for the 10 Hz and inactivated states, but possessed
reduced activity at the tonic state. Interestingly, inhibitory activity
6
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was gained for the other states for NaV1.2, leading to similar
potency for the two channels and no selectivity. Compound 16, in
which chloro groups replace the t-butyl groups in compound 2,
showed selectivity for the inactivated state of NaV1.2, and no
activity for NaV1.6. Compound 24, in which the phenolic group of
2 is absent, possessed no activity against either channel.
Strikingly, the benzyl derivative 17 showed activity against all
three states of the two NaV channels, with submicromolar potency
and some selectivity for the inactivated state of NaV1.2.
Compounds 18 and 26, which possess 4-acetamido or
sulfonamido groups, displayed similar profiles of inhibition against
only the inactivated states of NaV1.2 and NaV1.6; and similar
selectivities weakly in favour of NaV1.2. Finally, compound 25
bearing a 4-trifluoromethoxy group, showed a 10-fold or greater
selectivity for various states of NaV1.2, with a preference for
binding to the inactivated state of the channels.
[a] In some cases the selectivity could not be calculated.
Among the set of compounds studied, compounds 1 and 2
were selected on the basis of high NaV1.2 selectivity and high
lipophilicity for additional studies. In these studies we sought to
confirm binding using an alternative patch clamp protocol, and
also to examine the potential for slow binding. Protocol 2 was
performed on an IonWorks Barracuda instrument, following the
method of Kirsch (Figure 3b).[22] This protocol combines a 10 Hz
frequency-dependent voltage sequence with a long (2s) delay
with peak currents measured at -10 mV, and will enhance the
contribution of slow inactivated channels to the measured block.
To minimize the potential for variation between individual assays,
compound inhibition of NaV1.2 and NaV1.6 activities were
assessed on the same plate at the same time. Also included in
this analysis were positive controls, lamotrigine and lidocaine,
which are reported to modulate NaV channel fast inactivation.
Interestingly, compounds 1 and 2 both display reasonable
potencies for the inactivated state of Nav1.6. Collectively, these
data suggest that compounds 1 and 2 exhibit fast binding kinetics
to the inactivated state of Nav1.2, and slow binding kinetics for the
inactivated state of Nav1.6.
Table 4. Variation of Ar2 on NaV1.2 and NaV1.6 channel affinities (Protocol 1).
Block type
NaV1.2
IC50
NaV1.6
IC50
NaV1.6/
selectivity [a]
Ar1
(
µ
M)
( M)
µ
15
16
17
18
24
25
26
Tonic
10 Hz
>10
5.1
>10
4.8
-
Table 5. Affinities for binding to NaV1.2 and NaV1.6 channels primarily in the
slow inactivated state (Protocol 2).
0.9
Slow
inactivated
Type
inhibition
of
NaV1.2
NaV1.6
NaV1.2
selectivity[a]
2.2
2.2
1.0
IC50
(
µ
M)
IC50
(
µ
M)
Tonic
10 Hz
>10
>10
>10
>10
-
-
1
Tonic
10 Hz
Slow
18
13
5.1
>30
>30
23
-
-
Slow
inactivated
4.6
4.4
>10
-
inactivated
Tonic
10 Hz
3.3
1.8
5.2
2.9
1.6
1.6
2
Tonic
18.8
15.3
5.6
>30
>30
18
-
10 Hz
-
Slow
inactivated
Slow
3.2
0.5
2.0
4.0
inactivated
Tonic
10 Hz
>10
>10
>10
>10
-
-
lamotrigine
Tonic
10 Hz
>300
>300
94
>300
>300
50
-
-
Slow
inactivated
Slow
0.5
5.0
7.1
1.4
inactivated
Tonic
10 Hz
>10
>10
>10
>10
-
-
lidocaine
Tonic
10 Hz
2290
680
50
2150
565
75
~1
0.8
0.7
Slow
inactivated
Slow
>10
>10
-
inactivated
Tonic
10 Hz
12.5
3.6
137.8
56.3
11
16
[a] In some cases the selectivity could not be calculated.
Slow
inactivated
1.4
40.9
29
Tonic
10 Hz
>10
>10
>10
>10
-
-
Slow
inactivated
6.1
8.5
1.4
7
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10.1002/cmdc.201800781
ChemMedChem
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Metabolic stability and anti-epileptic testing of
compound 2
various states of each channel. While inhibition of the inactivated
state is common for existing NaV-blocking drugs, and is thought
to improve the safety margin by targeting the pathological state in
conditions such as epilepsy[26] and ischemia, new NaV-blocking
drugs targeting other disorders may be of utility in treating
conditions such as multiple sclerosis where demyelination of CNS
axons is present and amyotropic lateral sclerosis where Na+-
mediated hyperexcitability is involved.[26] Additionally, the low
lipophilicity of mexiletine results in its exclusion from the CNS and
consequent activity only on peripheral NaV channels. Our work
reveals more lipophilic variants that have the potential to cross the
blood-brain barrier and modulate NaV channels in the CNS.
Preliminary in vivo studies with 2 indicated that it provides
antiseizure actions in a mouse model of pharmaco-resistant
epilepsy in contrast to established sodium channel blockers such
as phenytoin and lamotrigine.[24] Compound 2 would be a
worthwhile candidate for detailed pharmacodynamic and
pharmacokinetic studies.
The metabolic stability of compounds 1 and 2 were assessed in
human liver microsomes in the presence of NADPH. According to
the data in Table 6, the compounds undergo high and
intermediate clearance levels. Preliminary identification of
metabolites by mass spectrometry revealed compound
undergoes O-dealkylation to the alcohol, N-dealkylation to
1
mexiletine, and mono and bis-oxygenation. Compound
underwent equivalent N-dealkylation, and monooxygenation.
2
Table 6. Metabolic evaluation of compounds
microsomes.[a]
1 and 2 in human liver
Cmpd
T1/2
(min)
CLint,
Predicted
CLint,
Predicted
CLblood
Predicted
EH
in
vitro
in vivo
(μL/min/
mg
(mL/min/kg)
(mL/min/kg)
protein)
1[b]
2
11
62
162
28
133
23
18
11
0.87
0.52
Experimental Section
The preparation of compounds HFI-1 (1), and 2-8 have been reported
previously.[27]
[a] Assays were performed with an NADPH regenerating system.
[b] Apparent minor non-NADPH mediated degradation was observed in metabolism
control samples (20 - 30% degradation over 60 min), however no putative metabolites
were detected. Predicted in vivo clearance parameters are reported; however, these
may be an underestimation of the true values.
General methods for reductive amination
Method A1
On the basis of its better stability relative to compound 1,
compound 2 was tested for antiepileptic activity in vivo in mice by
the Epilepsy Therapy Screening Program (ETSP) carried out by
the National Institute of Neurological Disorders and Stroke
(Bethesda, MD). Its current strategy has progressed beyond
using acute anticonvulsant screens such as the maximal
electroshock seizure and pentylenetetrazole tests to a mouse
screen of pharmaco-resistant epilepsy.[23] This is a low frequency
(6 Hz) electrical stimulation of brain at 22 mA for 3 seconds that
induces seizures reminiscent of ‘psychomotor seizures’ seen in
human limbic epilepsy. By increasing the current to 44 mA, the
conventional NaV blockers phenytoin and lamotrigine do not block
seizures so this is used as a model of therapy-resistant limbic
seizures.[24-25] Interestingly, intraperitoneal injection of compound
2 blocked these seizures and had a calculated ED50 of 49.9 ± 1.65
mg/kg. Separate groups of mice were injected with higher doses
of 2 and 6 hours later they were tested on a rota-rod apparatus to
measure any motor impairment. The dose causing 50% of mice
to fall off the rotating rod was calculated to be 90.5 ± 2.7 mg/kg.
Thus 2 is a potential candidate for treating pharmaco-resistant
epilepsy.
A mixture of amine (1 eq.), aldehyde (1.2 eq.) and MeOH (5 mL/mmol
amine) was stirred at room temperature under N2 until no more amine was
visible by thin-layer chromatography. NaBH4 (1-2 eq.) was added and the
mixture was stirred at r.t. for 30-60 min. The mixture was poured into sat.
aq. NaHCO3 and extracted with Et2O or EtOAc (3 times). The combined
organic extracts were dried (MgSO4), filtered, and the crude mixture
purified according to Protocol A or B (below) to obtain products as their
pure hydrochloride salts (see below).
Method A2
Where imine formation did not occur at room temperature in MeOH
according to Method A1 as assessed by thin-layer chromatography, the
reaction was instead conducted in EtOH at reflux. Upon consumption of
amine, the reaction mixture was cooled to r.t. prior to NaBH4 addition as
per Method A1.
Method B
A mixture of amine (1 eq.), aldehyde (1.2 eq.) and 1,2-dichloroethane (5-
10 mL/mmol amine) was stirred at room temperature under N2 for 1 h.
NaBH(OAc)3 (1.4 eq.) was added and the mixture was stirred for 2-4 h then
poured into sat. aq. NaHCO3and extracted with EtOAc. The organic phase
was washed sequentially with sat. aq. NaHCO3, H2O, then brine, dried
(MgSO4), filtered, and the crude solution of product treated in one of two
ways to obtain products as their pure hydrochloride salts (see below).
Owing to the tendency for this reaction to give tertiary amine products as
well as the desired hydrochloride salts, the crude material was preferably
purified using Purification Method B.
Conclusions
Here we report an examination of structure-activity relationships
for mexiletine against two neurologically significant sodium
channels, NaV1.2 and NaV1.6. Alkylation of the amine nitrogen
enhanced lipophilicity and dramatically enhanced potency for
NaV1.2 versus NaV1.6. Furthermore, conversion of the chiral
methylethylene linker to an achiral propylene linker did not
significantly affect potency. Further, modifications to the 2,6-
dimethylphenyl group resulted in a wide range of selectivities for
NaV1.2 versus NaV1.6, and a spectrum of activities across the
Purification Protocols
Protocol A
8
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10.1002/cmdc.201800781
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For reactions where very little starting amine remained the organic extract
was treated with 1 M HCl in MeOH affording a crystalline precipitate that
was collected by vacuum filtration. In cases where crystals did not form,
mixtures were evaporated and crystallized from either MeOH/Et2O,
MeOH/H2O, EtOH/H2O, EtOH/THF, MeCN/H2O or MeOH/THF, as noted.
If necessary compounds were purified by recrystallization using the
aforementioned solvent mixtures.
i) N-(3-(2,4,6-Trimethylphenoxy)propyl)phthalimide (30). A mixture of
2,4,6-trimethylphenol (2.66 g, 19.5 mmol), N-(3-bromopropyl)phthalimide
(5.00 g, 18.6 mmol), K2CO3 (2.70 g, 19.8 mmol) and DMF (20 mL) was
heated at 110 °C for 3 h. The mixture was concentrated under reduced
pressure then partitioned between H2O and dichloromethane (50 mL of
each). The organic layer was separated and washed sequentially with 0.3
M aq. NaOH (3 ´ 50 mL), H2O (50 mL), then sat. aq. NaCl (50 mL), dried
(MgSO4), filtered and concentrated under reduced pressure. The residue
was crystallized from hot methanol (25 mL) to afford the title compound as
crystals (2.90 g, 48%).
Protocol B
The dried organic extract was concentrated in vacuo and pure free-base
was purified by flash chromatography using either EtOAc/petroleum spirits
ii) 3-(2,4,6-Mesityloxy)propan-1-amine (31). A mixture of compound 30
(2.85 g, 8.65 mmol), hydrazine hydrate (3.53 g, 52 mmol) and ethanol (50
mL) was heated in an oil bath at 100 °C for 3 h under N2. After cooling to
r.t. the solvent was removed under reduced pressure and the residue
treated with 25% aq. NaOH. The mixture was extracted with EtOAc (3 ´
20 mL), the combined organic extracts were dried (MgSO4), concentrated
under reduced pressure and the residue purified by flash chromatography
(17:2:1 EtOAc/MeOH/H2O with 2 % Et3N) to give the title compound (1.47
g, 86%). 1H NMR (500 MHz, (CD3)2SO): d=1.78 (2 H, quintet, J 6.6 Hz,
CH2CH2N), 2.16 (6 H, s, 2 × CH3), 2.17 (3 H, s, CH3), 2.74 (2 H, t, J 6.8
Hz, CH2N), 3.72 (2 H, t, J 6.4 Hz, CH2O), 6.79 (2 H, s, Ar).
(10à100% with 1-2% Et3N) or EtOAc/MeOH/H2O/Et3N (97:2:1:2
à
7:2:1:0.2). After evaporation of the eluent the remaining Et3N was removed
by azeotroping with dichloromethane. The free base was converted to the
hydrochloride salt by either dissolving in Et2O and adding 1 M HCl in MeOH
or dissolving in MeOH and adding aq. HCl (either 1 M or 10 M). Where
necessary compounds were crystallized or recrystallized from MeOH/Et2O,
MeOH/H2O, EtOH/H2O, EtOH/THF, MeCN/H2O or MeOH/THF, as noted.
Preparation
of
4-(3-(3,5-di-tert-butyl-4-
hydroxybenzylamino)propyloxy)-3,5-dimethylbenzonitrile (9).
iii)
2,6-Di-tert-butyl-4-((3-(2,4,6-
i) 4-(3-Bromopropyloxy)-3,5-dimethylbenzonitrile (27). A mixture of
1,3-dibromopropane (683 mg, 3.39 mmol), 4-cyano-2,6-dimethylphenol
(100 mg, 0.679 mmol), Cs2CO3 (331 mg, 1.02 mmol) and acetone (20 mL)
was heated at reflux for 16 h. The mixture was filtered, concentrated under
reduced pressure and the residue purified by flash chromatography (20%
EtOAc/petroleum spirits) to afford the title compound (163 mg, 95%). 1H
NMR (400 MHz, CDCl3): d=2.30 (6 H, s, 2 ´ CH3), 2.33 (2 H, quint, J 6.2
Hz, CH2CH2N), 3.69 (2 H, t, J 6.2 Hz, CH2N), 3.93 (2 H, t, J 6.2 Hz, CH2O),
7.33 (2 H, d, J 0.5 Hz, Ar); 13C NMR (100 MHz, CD3OD): d=16.4, 30.1,
33.39, 69.55, 107.7, 119.2, 132.8, 132.9, 133.0, 159.6; HRMS-ESI m/z
calcd. for [C12H14BrNO+H]+: 268.0343, found: 268.0337.
mesityloxy)propylamino)methyl)phenol (10). Method A1 applied to
compound 31 and 3,5-di-tert-butyl-4-hydroxybenzylaldehyde followed by
purification by Protocol B (MeOH/H2O) afforded the title compound (494
mg, 71%). 1H NMR (500 MHz, CD3OD): d=1.44 (18 H, s, 2 ´ C(CH3)3),
2.18 (11 H, m, 3 ´ CH3,CH2CH2N), 3.32 (2 H, m, CH2N), 3.83 (2 H, t, J 5.8
Hz, CH2O), 4.17 (2 H, s, ArCH2N), 6.79 (2 H, s, Ar), 7.33 (2 H, s, Ar); 13
C
NMR (125 MHz, CD3OD): d=16.4, 20.8, 28.0, 30.6, 35.6, 46.1, 52.9, 70.2,
123.0, 128.0, 130.5, 131.2, 134.6, 140.0, 154.3, 156.6; HRMS-ESI m/z
calcd. for [C27H41NO2+H]+: 412.3215, found: 412.3228.
Preparation
of
2,6-di-tert-butyl-4-((3-(3,5-dimethylbiphenyl-4-
yloxy)propylamino) methyl)phenol (11).
ii) 4-(3-Azidopropyloxy)-3,5-dimethylbenzonitrile (28). A mixture of
compound 27 (163 mg, 0.648 mmol), NaN3 (46.3 mg, 0.711 mmol) and
DMSO (2 mL) was stirred overnight at r.t. Water (20 mL) was added and
the mixture was extracted with Et2O (2 ´ 50 mL). The combined organic
extracts were washed with H2O (50 mL), dried (MgSO4), filtered and
concentrated under reduced pressure to give the title compound (384 mg,
98%). 1H NMR (400 MHz, CDCl3): d=2.07 (2 H, quint, J 6.2 Hz, CH2CH2N),
2.29 (6 H, s, 2 ´ CH3), 3.61 (2 H, t, J 6.2 Hz, CH2N), 3.87 (2 H, t, J 6.2 Hz,
CH2O), 7.38 (2 H, s, Ar); 13C NMR (100 MHz, CDCl3): d=16.3, 29.8, 48.4,
69.4, 107.7, 119.1, 132.7, 133.0, 159.6; HRMS-ESI m/z calcd. for
[C12H14N4O+H]+: 231.1245, found: 231.1243.
i) N-(3-(3,5-Dimethylbiphenyl-4-yloxy)propyl)phthalimide (32).
A
mixture of 4-phenyl-2,6-dimethylphenol (351 mg, 1.77 mmol), N-(3-
bromopropyl)phthalimide (475 mg, 1.94 mmol), Cs2CO3 (1.15 g, 3.54
mmol) and acetone was heated at reflux for 16 h. The mixture was filtered
and concentrated under reduced pressure to give a thick oil, which was
purified by flash chromatography (10-15% EtOAc/petroleum spirits) gave
the title compound (566 mg, 83%). 1H NMR (400 MHz, CDCl3): d=2.23 (2
H, quint, J 6.2 Hz, CH2CH2N), 2.33 (6 H, s, 2 ´ CH3), 3.90, 3.98 (4 H, 2 ´
t, J 6.2 Hz, CH2N,CH2O), 7.22 (2 H, s, Ar), 7.27-7.32 (1 H, m, Ar), 7.37-
7.43 (2 H, m, Ar), 7.51-7.55 (2 H, m, Ar), 7.70-7.75 (2 H, m, Ar), 7.82-7.88
(2 H, m, Ar); 13C NMR (100 MHz, CD3OD): d=16.7, 29.6, 35.7, 70.0, 123.3,
126.9, 127.0, 127.7, 128.7, 131.2, 132.3, 134.0, 136.8, 141.0, 155.7,
168.4; HRMS-ESI m/z calcd. for [C25H23NO3+H]+: 386.1756, found:
386.1765.
iii) 4-(3-Aminopropyloxy)-3,5-dimethylbenzonitrile (29). A mixture of
Ph3P (200 mg, 0.763 mmol), compound 28 (384 mg, 0.630 mmol), water
(2 mL) and THF (10 mL) was stirred at r.t. overnight. The mixture was
poured into 10% aq. NaOH (30 mL) and extracted with EtOAc (2 ´ 20 mL).
The combined organic extracts were dried (MgSO4), filtered and
concentrated under reduced pressure. Flash chromatography (17:2:1
EtOAc/MeOH/H2O with 2% Et3N) gave the title compound (285 mg, 88%).
ii) 3-(3,5-Dimethylbiphenyl-4-yloxy)propan-1-amine (33). A mixture of
32 (546 mg, 1.41 mmol), hydrazine hydrate (578 mg, 8.5 mmol) and
ethanol (50 mL) was heated in an oil bath at 100 °C for 3 h under N2. After
cooling to r.t. the solvent was removed under reduced pressure and the
residue treated with 25% aq. NaOH. The mixture was extracted with EtOAc
iv)
4-(3-(3,5-Di-tert-butyl-4-hydroxybenzylamino)propyloxy)-3,5-
dimethylbenzonitrile (9). Method A1 applied to compound 29 and 3,5-di-
tert-butyl-4-hydroxybenzylaldehyde followed by purification Protocol B
(MeOH/H2O) afforded the title compound (74 mg, 34%). 1H NMR (400 MHz,
CD3OD): d=1.44 (18 H, s, 2 ´ C(CH3)3), 2.17-2.25 (2 H, m, CH2CH2N), 2.29
(6 H, s, 2 ´ CH3), 3.27-3.35 (2 H, obscured t, CH2N), 3.94 (2 H, t, J 6.0 Hz,
CH2O), 7.30 (2 H, s, Ar), 7.43 (2 H, s, Ar); 13C NMR (100 MHz, CD3OD):
d=16.3, 28.0, 30.6, 35.6, 45.8, 53.0, 70.4, 108.7, 119.7, 123.0, 128.0,
134.0, 134.1, 140.0, 156.6, 160.7; HRMS-ESI m/z calcd. for
[C27H38N2O2+H]+: 423.3011, found: 423.3008.
(3
´ 20 mL), the combined organic extracts were dried (MgSO4),
concentrated under reduced pressure and the residue purified by flash
chromatography (17:2:1 EtOAc/MeOH/H2O with 2 % Et3N) to give the title
compound (384 mg, 100%). 1H NMR (400 MHz, CD3OD): d=1.98 (2 H,
quint, J 6.2 Hz, CH2CH2N), 2.33 (6 H, s, 2 ´ CH3), 2.92 (2 H, t, J 6.2 Hz,
CH2N), 3.87 (2 H, t, J 6.2 Hz, CH2O), 7.23-7.28 (3 H, m, Ar), 7.34-7.40 (2
H, m, Ar), 7.49-7.54 (2 H, m, Ar); 13C NMR (100 MHz, CD3OD): d=16.8,
34.5, 40.1, 71.6, 127.7, 127.8, 128.4, 129.6, 132.1, 138.0, 142.2, 156.7;
HRMS-ESI m/z calcd. for [C17H21NO+H]+: 256.1701, found: 256.1704.
Preparation
of
2,6-Di-tert-butyl-4-((3-(2,4,6-
mesityloxy)propylamino)methyl)phenol (10).
9
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10.1002/cmdc.201800781
ChemMedChem
FULL PAPER
iii)
2,6-Di-tert-butyl-4-((3-(3,5-dimethylbiphenyl-4-
ii) 3-(2,4,6-Trimethylpyrimidin-5-yloxy)propan-1-amine (37). Prepared
from 36 and purification, as per 33, afforded the title compound (82.7 mg,
94%). 1H NMR (400 MHz, CDCl3): d=2.04 (2 H, m, CH2CH2O), 2.41 (6 H,
s, 2 ´ CH3), 2.59 (3 H, s, CH3), 3.06 (2 H, t, J 7.0 Hz, CH2N), 3.85 (2 H, t,
J 6.0 Hz, CH2O); HRMS-ESI m/z calcd. for [C10H17N3O+H]+: 196.1444,
found: 196.1442.
yloxy)propylamino)methyl)phenol (11). Method A1 applied to 33 and
3,5-di-tert-butyl-4-hydroxybenzylaldehyde followed by purification
according to Protocol B (MeOH/H2O) afforded the title compound (210 mg,
27%). 1H NMR (500 MHz, CD3OD): d=1.45 (18 H, s, 2 ´ C(CH3)3), 2.22 (2
H, m, CH2CH2N), 2.30 (6 H, s, 2 ´ CH3), 3.35 (2 H, m, CH2N), 3.83 (2 H, t,
J 5.8 Hz, CH2O), 4.18 (2 H, s, ArCH2N), 7.23-7.32 (5 H, m, Ar), 7.39 (2 H,
t, J 7.6 Hz, Ar), 7.53 (2 H, t, J 7.8 Hz); 13C NMR (100 MHz, CD3OD): d=16.6,
28.1, 46.1, 53.0, 70.2, 123.0, 127.8, 128.0, 128.5, 129.7, 132.1, 138.5,
140.1, 142.1, 156.2, 156.6; HRMS-ESI m/z calcd. for [C32H43NO2+H]+:
474.3372, found: 474.3367.
iii)
2,6-Di-tert-butyl-4-((3-(2,4,6-trimethylpyrimidin-5-
yloxy)propylamino)methyl) phenol (13). Method A1 applied to 37 and
3,5-di-tert-butyl-4-hydroxybenzylaldehyde followed by purification Protocol
B (MeOH/Et2O) afforded the title compound (65.2 mg, 59%). 1H NMR (400
MHz, CD3OD): d=1.45 (18 H, s, 2 ´ C(CH3)3), 2.34-2.37 (2 H, m, CH2CH2N),
2.68 (6 H, s, 2 ´ ArCH3), 2.79 (3 H, s, ArCH3), 3.31-3.36 (2 H, obscured m,
CH2N), 4.15 (2 H, t, J 6.0 Hz, CH2O), 4.18 (2 H, s, ArCH2N), 7.35 (2 H, s,
Ar); HRMS-ESI m/z calcd. for [C25H39N3O2+H]+: 414.3115, found:
414.3115.
Preparation
of
2,6-di-tert-butyl-4-((3-(2,3,6-
trimethylphenoxy)propylamino)methyl) phenol (12).
i) N-(3-(2,3,6-Trimethylphenoxy)propyl)phthalimide (34). A mixture of
2,3,6-trimethylphenol (990 mg, 7.27 mmol), N-(3-bromopropyl)phthalimide
(2.14 g, 8.00 mmol), K2CO3 (3.01 g, 21.8 mmol) and DMF (5 mL) was
heated at 70 °C for 2 h. The mixture was concentrated under reduced
pressure then partitioned between EtOAc and sat. aq. NaHCO3 (50 mL of
each). The organic layer was separated and the aqueous layer extracted
with EtOAc (2 ´ 20 mL). The combined organic extracts were washed with
sat. aq. NaHCO3 (50 mL) then sat. aq. NaCl (50 mL), dried (MgSO4),
filtered and concentrated under reduced pressure. The residue was
purified by flash chromatography (10-15% EtOAc/petroleum spirits) to give
the title compound (1.01 g, 43%). 1H NMR (400 MHz, CDCl3): d=2.17-2.24
(11 H, m, 3 ´ CH3,CH2CH2O), 3.82 (2 H, t, J 6.3 Hz, CH2N), 3.96 (2 H, t, J
7.3 Hz, CH2O), 6.81 (1 H, d, J 7.6 Hz, Ar), 6.89 (1 H, d, J 7.6 Hz, Ar), 7.72
(2 H, td, J 5.8, 2.7 Hz, Ar), 7.84-7.88 (2 H, m, Ar); 13C NMR (100 MHz,
CDCl3): d=12.5, 16.3, 19.8, 29.4, 35.6, 69.9, 123.1, 125.1, 127.8, 127.9,
129.4, 132.1, 133.8, 135.7, 155.7, 168.2; HRMS-ESI m/z calcd. for
[C20H21NO3+H]+: 324.1594, found: 324.1588.
Preparation
of
2,6-di-tert-butyl-4-((3-(2-isopropyl-4,6-
dimethylpyrimidin-5-yloxy) propylamino) methyl)phenol (14).
i)
N-(3-(2-Isopropyl-4,6-dimethylpyrimidin-5-
yloxy)propyl)phthalimide (38) Prepared from 2-isopropyl-4,6-
dimethylpyrimidinol and N-(3-bromopropyl)phthalimide, as per 32, and
purification by flash chromatography (20-30% EtOAc/petroleum spirits)
gave the title compound (422 mg 86%). 1H NMR (400 MHz, CDCl3): d=1.28
(6 H, d, J 7.0 Hz, CH(CH3)2), 2.21 (2 H, quintet, J 6.8 Hz, CH2CH2N), 2.45
(6 H, s, 2 ´ ArCH3), 3.11 (1 H, septet, J 7.0 Hz, CH(CH3)2), 3.87, 3.95 (4
H, 2 ´ t, J 6.2 Hz, CH2N,CH2O), 7.71-7.76, 7.84-7.89 (4 H, 2 ´ m, Ar);13
NMR (100 MHz, CDCl3): d=19.5, 22.4, 29.9, 35.8, 37.5, 71.2, 123.8, 132.6,
134.5, 148.8, 159.9, 168.8, 169.9; HRMS-ESI m/z calcd. for
[C20H23N3O3+H]+: 354.1818, found: 354.1813.
C
ii) 3-(2-Isopropyl-4,6-dimethylpyrimidin-5-yloxy)propan-1-amine (39).
Prepared from 38 and purification, as per 33, gave the title compound (245
mg, 94%). 1H NMR (400 MHz, CD3OD): d=1.26 (6 H, d, J 7.0 Hz,
CH(CH3)2), 1.96 (2 H, quintet, J 6.8 Hz, CH2CH2N), 2.44 (6 H, s, 2 ´ ArCH3),
3.06 (1 H, septet, J 7.0 Hz, CH(CH3)2), 3.28 (2 H, t, J 6.2 Hz, CH2N), 3.90
(4 H, t, J 6.2 Hz, CH2O);13C NMR (100 MHz, CD3OD): d=18.8, 22.2, 34.2,
37.9, 39.7, 72.6, 149.8, 161.4, 170.3; HRMS-ESI m/z calcd. for
[C12H21N3O+H]+: 224.1757, found: 244.1763.
ii) 3-(2,3,6-Trimethylphenoxy)propan-1-amine (35). Prepared from 34
and purification, as described for 33, afforded the title compound (513 mg,
85%). 1H NMR (400 MHz, CDCl3): d=1.95 (2 H, quintet, J 6.6 Hz,
CH2CH2O), 2.18 (3 H, s, CH3), 2.23 (3 H, s, CH3), 2.25 (3 H, s, CH3), 2.99
(2 H, t, J 6.9 Hz, CH2N), 3.80 (2 H, t, J 6.2 Hz, CH2O), 6.82 (1 H, d, J 7.6
Hz, Ar), 6.90 (1 H, d, J 7.6 Hz, Ar); 13C NMR (100 MHz, CDCl3): d=12.5,
16.3, 20.0, 34.3, 39.7, 70.5, 125.2, 127.9, 128.1, 129.5, 135.8, 155.8;
HRMS-ESI m/z calcd. for [C12H19NO+H]+: 194.1539, found: 194.1541.
iii)
2,6-Di-tert-butyl-4-((3-(2-isopropyl-4,6-dimethylpyrimidin-5-
iii)
2,6-Di-tert-butyl-4-((3-(2,3,6-
yloxy)propylamino) methyl)phenol (14). Method A1 applied to 39 and
3,5-di-tert-butyl-4-hydroxybenzylaldehyde, followed by purification
Protocol B (MeOH/H2O) afforded the title compound (306 mg, 57%). 1H
NMR (500 MHz, CD3OD): d=1.40 (6 H, d, J 6.8 Hz, CH(CH3)2), 1.45 (18 H,
s, 2 ´ C(CH3)3), 2.27-2.32 (2 H, m, CH2CH2N), 2.68 (6 H, s, 2 ´ ArCH3),
3.22-3.28 (3 H, obscured multiplet, CH(CH3)2,CH2N), 4.14 (2 H, t, J 6.0 Hz,
CH2O), 4.18 (2 H, s, ArCH2N), 7.34 (2 H, s, Ar); 13C NMR (125 MHz,
CD3OD): d=18.3, 21.3, 27.9, 30.7, 35.3, 35.7, 45.4, 53.1, 72.6, 123.1,
128.0, 140.0, 150.3, 156.6, 165.5; HRMS-ESI m/z calcd. for
[C27H43N3O2+H]+: 442.3433, found: 442.3433.
trimethylphenoxy)propylamino)methyl)phenol (12). Method A1
applied to 35 and 3,5-di-tert-butyl-4-hydroxybenzylaldehyde followed by
purification Protocol A (MeOH/H2O) afforded the title compound (244 mg,
58%). 1H NMR (400 MHz, CD3OD): d=1.44 (18 H, s, 2 ´ C(CH3)3), 2.14 (3
H, s, CH3), 2.18-2.26 (8 H, m, 2 ´ CH3,CH2CH2N), 3.33-3.36 (2 H, m,
CH2N), 3.81 (2 H, t, J 5.8 Hz, CH2O), 4.18 (2 H, s, ArCH2N), 6.80 (1 H, d,
J 7.7 Hz, Ar), 6.87 (1 H, d, J 7.7 Hz, Ar), 7.34 (2 H, s, Ar); 13C NMR (100
MHz, CD3OD): d=12.6, 16.5, 19.9, 28.0, 30.6, 35.6, 46.1, 52.9, 70.3, 123.0,
126.6, 128.0, 128.8, 129.0, 130.1, 137.0, 140.0, 156.3, 156.6; HRMS-ESI
m/z calcd. for [C27H41NO2+H]+: 412.3210, found: 412.3211.
4-((3-(2,6-Dimethylphenoxy)propylamino)methyl)phenol (15). Method
A1 applied to 3-(2,6-dimethylphenoxy)propylamine[28] and 4-
hydroxybenzaldehyde, followed by purification Protocol B (MeOH/H2O)
afforded the title compound (156 mg, 42%). 1H NMR (500 MHz, CD3OD):
d=2.19-2.25 (8 H, m, 2 ´ CH3,CH2CH2N), 3.33-3.36 (2 H, obscured m,
CH2N), 3.88 (2 H, t, J 5.9 Hz, CH2O), 4.19 (2 H, s, ArCH2N), 6.87-6.93 (3
H, m, Ar), 7.01 (2 H, d, J 7.4 Hz, Ar), 7.36-7.38 (2 H, m, Ar); 13C NMR (125
MHz, CD3OD): d=16.4, 28.1, 46.1, 52.1, 70.0, 116.9, 122.9, 125.3, 130.0,
131.7, 132.6, 156.6, 160.0; HRMS-ESI m/z calcd. for [C18H23NO2+H]+:
286.1807, found: 286.1804.
Preparation
yloxy)propylamino) methyl) phenol (13).
of
2,6-di-tert-butyl-4-((3-(2,4,6-trimethylpyrimidin-5-
i) N-(3-(2,4,6-Trimethylpyrimidin-5-yloxy)propyl)phthalimide (36).
Prepared
from
2,4,6-trimethylpyrimidinol
and
N-(3-
bromopropyl)phthalimide as described for 34, and purification by flash
chromatography (7:3:0.1 EtOAc/petroleum spirits/Et3N) gave the title
compound (301.9 mg, 85%). 1H NMR (400 MHz, CDCl3): d=2.16 (2 H,
quintet, J 6.8 Hz, CH2CH2O), 2.39 (6 H, s, 2 ´ CH3), 2.55 (3 H, s, CH3),
3.82 (2 H, t, J 6.2 Hz, CH2N), 3.90 (2 H, t, J 7.1 Hz, CH2O), 7.66-7.70 (2
H, m, Ar), 7.78-7.83 (2 H, m, Ar); 13C NMR (100 MHz, CDCl3): d=19.0, 25.3,
29.5, 35.3, 70.9, 123.4, 132.1, 134.1, 148.4, 159.6, 162.0, 168.3; HRMS-
ESI m/z calcd. for [C18H19N3O3+H]+: 326.1499, found: 326.1501.
2,6-Dichloro-4-((3-(2,6-dimethylphenoxy)propylamino)methyl)phenol
(16). Method A1 applied to 3-(2,6-dimethylphenoxy)propylamine[28] and
3,5-dichloro-4-hydroxybenzaldehyde, followed by purification using
Protocol B (MeOH/H2O) afforded the title compound (318 mg, 64%). 1H
10
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10.1002/cmdc.201800781
ChemMedChem
FULL PAPER
NMR (400 MHz, CD3OD): d=2.19-2.24 (8 H, m, 2 ´ CH3,CH2CH2N), 3.33-
3.35 (2 H, m, CH2N), 3.87 (2 H, t, J 5.9 Hz, CH2O), 4.19 (2 H, s, ArCH2N),
6.89 (1 H, t, J 7.5 Hz, Ar), 6.98-7.00 (2 H, m, Ar), 7.53 (2 H, s, Ar); 13C
NMR (100 MHz, CD3OD): d=16.4, 28.1, 46.4, 50.9, 69.9, 123.82, 123.84,
124.8, 125.2, 130.0, 131.5, 131.7, 152.0, 156.6; HRMS-ESI m/z calcd. for
[C18H21Cl2NO2+H]+: 354.1022, found: 354.1027.
and 3,5-di-tert-butyl-4-hydroxybenzylaldehyde, followed by purification
Protocol B (MeCN/H2O) afforded the title compound (68 mg, 21%). 1H
NMR (500 MHz, CD3OD): d=1.45 (18 H, s, 2 ´ C(CH3)3), 2.19-2.25 (2 H,
m, CH2CH2N), 2.27 (6 H, s, 2 ´ CH3), 3.32-3.36 (2 H, m, CH2N), 3.90 (2 H,
t, J 5.8 Hz, CH2O), 4.18 (2 H, s, ArCH2N), 7.05 (1 H, dd, J 5.1, 3.6 Hz, Ar),
7.26-7.32 (6 H, m, Ar); 13C NMR (125 MHz, CD3OD): d=16.5, 28.1, 30.6,
35.6, 46.1, 53.0, 70.2, 123.0, 123.8, 125.4, 127.4, 127.9, 129.0, 131.9,
132.4, 140.1, 145.1, 156.2, 156.7; HRMS-ESI m/z calcd. for
[C30H41NO2S+H]+: 490.2936, found: 480.2929.
N-Benzyl-3-(2,6-dimethylphenoxy)propan-1-amine (17). Method A1
applied to 3-(2,6-dimethylphenoxy)propylamine[28] and benzaldehyde,
followed by purification using Protocol B (MeOH/H2O) afforded the title
compound (135 mg, 37%). 1H NMR (500 MHz, CD3OD): d=2.16-2.22 (2 H,
m, CH2CH2N), 2.23 (6 H, 2 ´ CH3), 3.34 (2 H, t, J 6.4 Hz, CH2N), 3.87 (2
H, t, J 5.8 Hz, CH2O), 4.26 (2 H, s, ArCH2N), 6.90 (1 H, t, J 6.0 Hz, Ar),
7.00 (2 H, m, Ar), 7.45-7.53 (5 H, m, Ar); 13C NMR (125 MHz, CD3OD):
d=16.4, 28.1, 46.5, 52.4, 70.0, 125.3, 130.0, 130.3, 130.7, 131.1, 131.7,
132.5, 156.6; HRMS-ESI m/z calcd. for [C18H23NO+H]+: 270.1857, found:
270.1857.
Preparation of 2,6-di-tert-butyl-4-((3-(2,6-dimethyl-4-(thiophen-3-
yl))propylamino) methyl)phenol (20).
i) 2,6-Dimethyl-4-(thiophen-3-yl)phenol (43). Pd(dppt)Cl2 was added to
a degassed (N2) mixture of 3,5-dimethyl-4-hydroxyphenyl boronic acid
pinacol ester (1.00 g, 4.0 mmol), 2-bromothiophene (853 mg, 5.20 mmol),
K3PO4 (1.80 g, 8.48 mmol) and dry DMF (30 mL). The mixture was heated
at 110 °C with stirring for 16 h, cooled to r.t and then concentrated under
reduced pressure. The residue was partitioned between EtOAc and 1M aq.
HCl, the organic layer was separated, dried (MgSO4), filtered and
concentrated under reduced pressure. The crude material was crystallized
from petroleum spirits to give the title compound (373 mg, 45%). 1H NMR
(400 MHz, CDCl3): d=2.19 (6 H, s, 2 ´ CH3), 7.20-7.26 (5 H, m, Ar); 13C
NMR (100 MHz, CDCl3): d=16.1, 118.8, 123.4, 126.0, 126.4, 126.9, 128.4,
142.4, 151.7; HRMS-ESI m/z calcd. for [C12H12OS+H]+: 205.0687, found:
205.0684.
N-(4-((3-(2,6-
Dimethylphenoxy)propylamino)methyl)phenyl)acetamide
(18).
Method
B
applied to 3-(2,6-dimethylphenoxy)propylamine[28] and 4-
acetamidobenzaldehyde, followed by purification Protocol B (MeOH/Et2O)
afforded the title compound (82.2 mg, 45%). 1H NMR (500 MHz, CD3OD):
d=2.14 (3 H, NHAc), 2.18-2.24 (8 H, m, 2 ´ CH3,CH2CH2N), 3.35 (2 H, t, J
7.9 Hz, CH2N), 3.87 (2 H, t, J 5.8 Hz, CH2O), 4.24 (2 H, s, ArCH2N), 6.90
(1 H, t, J 7.5 Hz, Ar), 7.00 (2 H, d, J 7.5 Hz, Ar), 7.47 (2 H, d, J 8.5 Hz, Ar),
7.68 (2 H, d, J 8.5, Hz Ar); 13C NMR (100 MHz, CD3OD): d=16.42, 16.43,
23.89, 23.91, 28.1, 46.3, 52.0, 70.0, 121.4, 125.3, 127.6, 130.0, 131.69,
131.73, 141.4, 156.6, 171.8; HRMS-ESI m/z calcd. for [C20H26N2O2+H]+:
327.2067, found: 327.2068.
ii) N-(3-(2,6-Dimethyl-4-(thiophen-3-yl)phenoxy)propyl)phthalimide
(44). Prepared from 43, as per 32, and purification by flash
chromatography (15% EtOAc/petroleum spirits) afforded the title
compound (309 mg, 96%). 1H NMR (400 MHz, CDCl3): d=2.22 (2 H, quintet,
J 6.8 Hz, CH2CH2N), 2.31 (6 H, s, 2 ´ CH3), 3.88, 3.95 (4 H, 2 ´ t, J 6.2 Hz,
CH2N,CH2O), 7.02-7.06 (1 H, m, Ar), 7.18-7.25 (4 H, m, Ar), 7.70-7.74,
7.84-7.88 (4 H, 2 ´ m, Ar); 13C NMR (100 MHz, CD3OD): d=16.6, 29.6,
35.7, 70.0, 119.6, 123.2, 126.0, 126.5, 127.0, 131.2, 131.5, 132.2, 134.0,
142.2, 155.4, 168.5.
Preparation of 2,6-di-tert-butyl-4-((3-(2,6-dimethyl-4-(thiophen-2-
yl)phenoxy) propylamino)methyl)phenol (19).
i) 2,6-Dimethyl-4-(thiophen-2-yl)phenol (40). Pd(dppt)Cl2 was added to
a degassed (N2) mixture of 3,5-dimethyl-4-hydroxyphenyl boronic acid
pinacol ester (1.00 g, 4.0 mmol), 2-bromothiophene (853 mg, 5.20 mmol),
K3PO4 (1.8 g, 8.48 mmol) and dry DMF (30 mL). The mixture was heated
at 100 °C with stirring for 16 h, filtered through a silica plug rinsed with
EtOAc, and the filtrate was concentrated under reduced pressure. The
residue was purified by flash chromatography (10% EtOAc/petroleum
spirits) to give the title compound. 1H NMR (400 MHz, CDCl3): d=2.25 (6
H, s, 2 ´ CH3), 7.00 (1 H, m, Ar), 7.13-7.18 (2 H, m, Ar), 7.21 (2 H, s, Ar);
13C NMR (100 MHz, CDCl3): d=16.1, 122.00, 123.6, 123.7, 126.6, 126.9,
126.9, 128.0, 144.8, 152.1; HRMS-ESI m/z calcd. for [C12H12OS+H]+:
205.0687, found: 205.0684.
iii) 3-(2,6-Dimethyl-4-(thiophen-3-yl)phenoxy)propan-1-amine (45).
Prepared from 44 and purification, as per 32, afforded the title compound
(123 mg, 91%). 1H NMR (400 MHz, CD3OD): d=1.98 (2 H, quintet, J 6.8
Hz, CH2CH2N), 2.29 (6 H, s, 2 ´ CH3), 2.94 (2 H, t, J 6.2 Hz, CH2N), 3.85
(2 H, t, J 6.2 Hz, CH2O), 7.34 (2 H, s, Ar), 7.36-7.48 (3 H, m, Ar); 13C NMR
(100 MHz, CD3OD): d=16.5, 33.8, 40.0, 71.4, 120.4, 127.0, 127.3, 127.8,
132.1, 132.9, 143.4, 157.3; HRMS-ESI m/z calcd. for [C15H19NOS+H]+:
262.1266, found: 262.1261.
iv)
2,6-Di-tert-butyl-4-((3-(2,6-dimethyl-4-(thiophen-3-
ii) N-(3-(2,6-Dimethyl-4-(thiophen-2-yl)phenoxy)propyl)phthalimide
(41). Prepared from 40, as per 32, and purification by flash
chromatography (15-20% EtOAc/petroleum spirits) gave the title
compound (320 mg, 92%). 1H NMR (400 MHz, CDCl3): d=2.12 (2 H, quintet,
J 6.8 Hz, CH2CH2N), 2.30 (6 H, s, 2 ´ CH3), 3.87, 3.97 (4 H, 2 ´ t, J 6.2 Hz,
CH2N,CH2O), 7.02-7.06 (1 H, m, Ar), 7.18-7.25 (4 H, m, Ar), 7.70-7.74,
7.84-7.88 (4 H, 2 ´ m, Ar);13C NMR (100 MHz, CD3OD): d=16.6, 29.6, 35.7,
70.0, 122.6, 123.4, 124.3, 126.6, 128.0, 130.1, 131.3, 132.3, 134.1, 144.4,
155.8, 168.5.
yl)phenoxy)propylamino)methyl) phenol (20). Method A1 applied to 45
and 3,5-di-tert-butyl-4-hydroxybenzylaldehyde, followed by purification
Protocol B (MeOH/H2O) afforded the title compound (105 mg, 49%). 1H
NMR (500 MHz, CD3OD): d=1.45 (18 H, s, 2 ´ C(CH3)3), 2.19-2.25 (2 H,
m, CH2CH2N), 2.28 (6 H, s, 2 ´ CH3), 3.35 (2 H, m, CH2N), 3.90 (2 H, t, J
5.8 Hz, CH2O), 4.18 (2 H, s, ArCH2N), 7.32 (4 H, d, J 8.7 Hz, Ar), 7.37-
7.38 (1 H, m, Ar), 7.43 (1 H, dd, J 5.0, 3.0 Hz, Ar), 7.49 (1 H, dd, J 2.9, 1.3,
Ar); 13C NMR (400 MHz, CD3OD): d=16.7, 28.0, 30.6, 35.6, 46.0, 52.9,
70.2, 120.5, 123.0, 127.0, 127.1, 127.8, 128.0, 132.0, 133.2, 139.9, 143.1,
155.8, 156.5; HRMS-ESI m/z calcd. for [C30H41NO2S+H]+: 480.2936,
found: 480.2945.
iii) 3-(2,6-Dimethyl-4-(thiophen-2-yl)phenoxy)propan-1-amine (42).
Prepared from 41 and purification, as per 33, gave the title compound (123
mg, 91%). 1H NMR (400 MHz, CD3OD): d=1.99 (2 H, quintet, J 6.8 Hz,
CH2CH2N), 2.30 (6 H, s, 2 ´ CH3), 2.95 (2 H, t, J 6.2 Hz, CH2N), 3.87 (2 H,
t, J 6.2 Hz, CH2O), 7.04 (1 H, m, Ar), 7.25-7.34 (4 H, m, Ar);13C NMR (100
MHz, CD3OD): d=16.4, 33.1, 39.3, 70.3, 122.6, 124.3, 126.6, 128.0, 130.1,
131.4, 144.3, 155.5; HRMS-ESI m/z calcd. for [C15H19NOS+H]+: 262.1266,
found: 262.1263.
Preparation
of
2,6-Di-tert-butyl-4-((3-(3,5-dimethyl-1H-pyrazol-4-
yloxy)propylamino) methyl)phenol (21).
i) N-(3-(3,5-Dimethyl-1H-pyrazol-4-yloxy)propyl)phthalimide (46).
Prepared from N-(3-bromopropyl)phthalimide and 3,5-dimethyl-1H-
pyrazol-4-ol,[29] as per 32, and purification by flash chromatography (90%
EtOAc/petroleum spirits) afforded the title compound (290 mg, 25%). 1H
NMR (400 MHz, CDCl3): d=2.07 (2 H, dd, J 13.6, 6.4 Hz, CH2CH2N), 2.19
(6 H, s, 2 ´ CH3), 3.86 (4 H, td, J 6.6, 4.7 Hz, CH2N,CH2O), 7.67 (2 H, dd,
iv)
2,6-Di-tert-butyl-4-((3-(2,6-dimethyl-4-(thiophen-2-
yl)phenoxy)propylamino)methyl) phenol (19). Method A1 applied to 42
11
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10.1002/cmdc.201800781
ChemMedChem
FULL PAPER
J 5.4, 3.1, Ar), 7.81 (2 H, dd, J 5.5, 3.1 Hz, Ar); 13C NMR (100 MHz, CDCl3):
d=9.8, 29.2, 35.4, 72.0, 123.2, 132.1, 140.0, 134.6, 138.9, 168.4; HRMS-
ESI m/z calcd. for [C16H17N3O3+H]+: 300.1343, found: 300.1341.
3,5-di-tert-butyl-4-hydroxybenzylaldehyde, followed by purification by
Protocol A (MeOH/H2O) afforded the title compound (98.2 mg, 34%). 1H
NMR (400 MHz, CD3OD): d=1.44 (18 H, s, 2 ´ C(CH3)3), 2.16-2.18 (2 H,
m, CH2CH2N), 2.29 (3 H, s, NCCH3), 2.31 (3 H, s, NCCH3), 3.25-3.29 (2
H, m, CH2N), 3.83 (3 H, s, NCH3), 4.03 (2 H, t, J 5.3 Hz, CH2O), 4.16 (2 H,
s, ArCH2N), 7.33 (2 H, s, Ar); 13C NMR (100 MHz, CD3OD): d=8.5, 9.1,
27.8, 30.6, 35.6, 36.1, 45.7, 53.1, 73.6, 123.1, 128.0, 137.4, 137.9, 140.0,
140.4, 156.6; HRMS-ESI m/z calcd. for [C24H38N3O2+H]+: 402.3115, found:
402.3115.
ii) 3-(3,5-Dimethyl-1H-pyrazol-4-yloxy)propan-1-amine (47). A mixture
of the phthalimide 46 (192 mg, 0.644 mmol), hydrazine hydrate (193 mg,
3.86 mmol) and ethanol (11 mL) was heated in an oil bath at 100 °C for 16
h under N2. After cooling to 0 °C the mixture was filtered and the filtrate
was concentrated under reduced pressure. The residue was triturated with
14:5:1 CH2Cl2/MeOH/H2O, the liquid fraction was concentrated to give
enriched (~90% pure by 1H NMR) amine, which was used in subsequent
reactions without further purification (94.8 mg, 87%). 1H NMR (500 MHz,
CD3OD): d=1.85 (2 H, quint, J 7.0 Hz, CH2CH2N), 2.15 (6 H, s, 2 ´ CH3),
2.85 (2 H, t, J 7.0 Hz, CH2N), 3.88 (2 H, t, CH2O); 13C NMR (125 MHz,
CD3OD): d=9.6, 33.9, 39.8, 73.9, 135.6 (2 C, br s), 140.1; HRMS-ESI m/z
calcd. for [C8H15N3O+H]+: 170.1288, found: 170.1301.
Preparation
of
2,6-Di-tert-butyl-4-((3-(2,4,6-
trichlorophenoxy)propylamino)methyl) phenol (23).
i) N-(3-(2,4,6-Trichlorophenoxy)propyl)phthalimide (51). Prepared
from N-(3-bromopropyl)phthalimide and 2,4,6-trichlorophenol, as per 32,
and purification by crystallization from EtOH afforded the title compound
(1.84 g, 94%). 1H NMR (400 MHz, CDCl3): d=2.19-2.26 (2 H, m, CH2CH2N),
3.96, 4.07 (4 H, 2 × t, J 6.5 Hz, CH2N,CH2O), 7.26 (2 H, s, Ar), 7.68-7.72
(2 H, m, Ar), 7.83 (2 H, m, Ar); 13C NMR (100 MHz, CDCl3): d=29.2, 35.5,
71.5, 123.4, 128.8, 129.6, 130.1, 132.2, 134.0, 150.5, 168.4; HRMS-ESI
m/z calcd. for [C17H12Cl3NO3+H]+: 385.9932, found: 385.9925.
iii)
2,6-Di-tert-butyl-4-((3-(3,5-dimethyl-1H-pyrazol-4-
yloxy)propylamino)methyl)phenol (21). Method A1 applied to 47 and
3,5-di-tert-butyl-4-hydroxybenzylaldehyde, followed by purification by
Protocol A (MeOH/Et2O) afforded the title compound (210 mg, 85%). 1H
NMR (400 MHz, CD3OD): d=1.45 (18 H, s, 2 ´ C(CH3)3), 2.16-2.22 (2 H,
m, CH2CH2N), 2.35 (6 H, s, 2 ´ NCCH3), 3.26-3.30 (2 H, m, CH2N), 4.08
(2 H, t, J 6.0 Hz, CH2O), 4.17 (2 H, s, ArCH2N), 7.35 (2 H, s, Ar); 13C NMR
(100 MHz, CD3OD): d=8.8, 27.8, 30.6, 35.6, 45.6, 53.0, 73.2, 123.1, 128.0,
137.7, 140.0, 156.6; HRMS-ESI m/z calcd. for [C23H36N3O2+H]+: 388.2959,
found: 388.2965).
ii) 3-(2,4,6-Trichlorophenoxy)propan-1-amine (52).
Prepared from 51, as per 31, and purification by flash chromatography
(17:2:1:0.4 EtOAc/MeOH/H2O/Et3N) gave the title compound (1.03 g,
91%). 1H NMR (500 MHz, CDCl3): d=1.96-2.01 (2 H, m, CH2CH2N), 2.92
(2 H, t, J 7.0 Hz, CH2N), 4.09 (2 H, t, J 6.0 Hz, CH2O), 7.45 (2 H, d, J 0.6
Hz, Ar); 13C NMR (125 MHz, CDCl3): d=34.0, 39.8, 73.1, 130.0, 130.8,
131.2, 151.9; HRMS-ESI m/z calcd. for [C9H10Cl3NO+H]+: 255.9877,
found: 255.9880.
Preparation of 2,6-Di-tert-butyl-4-((3-(1,3,5-trimethyl-1H-pyrazol-4-
yloxy)propylamino) methyl)phenol (22).
i) 4-(3-Bromopropoxy)-1,3,5-trimethyl-1H-pyrazole (48). A mixture of
1,3,5-trimethyl-1H-pyrazole
(320
mg,
2.91
mmol),
N-(3-
iii)
2,6-Di-tert-butyl-4-((3-(2,4,6-
bromopropyl)phthalimide (587 mg, 2.91 mmol), K2CO3 (2.01 g, 14.3 mmol)
and DMF (30 mL) was heated at 60 °C for 4 h. The mixture was cooled to
r.t., filtered, concentrated under reduced pressure and partitioned between
EtOAc and sat. aq. NaHCO3 (20 mL of each). The organic layer was
separated and the aqueous layer extracted with EtOAc (2 ´ 10 mL). The
combined organic extracts were sequentially washed with sat. aq.
NaHCO3 (20 mL), H2O (20 mL), then sat. aq. NaCl (20 mL), dried (MgSO4),
filtered and concentrated under reduced pressure. The residue was
purified by flash chromatography (4:4:1à3:5:1 toluene/CH2Cl2/acetone) to
give the title compound (226 mg, 31%). 1H NMR (500 MHz, CDCl3): d=2.08
(3 H, s, CH3), 2.10 (3 H, s, CH3), 2.13 (2 H, CH2CH2N), 3.55 (2 H, t, J 6.0
Hz, CH2Br), 3.57 (3 H, s, NCH3), 3.83 (2 H, t, J 6.0 Hz, CH2O); 13C NMR
(125 MHz, CDCl3): d=8.4, 10.7, 30.0, 32.9, 36.2, 128.9, 138.2, 138.7;
HRMS-ESI m/z calcd. for [C9H15BrN2O+H]+: 247.0441, found: 247.0441.
trichlorophenoxy)propylamino)methyl)phenol (23). Method A1 applied
to 51 and 3,5-di-tert-butyl-4-hydroxybenzylaldehyde, followed by
purification Protocol B (MeOH/Et2O) afforded the title compound (580 mg,
97%). 1H NMR (500 MHz, CD3OD): d=1.44 (18 H, s, 2 ´ C(CH3)3), 2.23-
2.28 (2 H, m, CH2CH2N), 3.34-3.37 (2 H, m, CH2N), 4.12 (2 H, t, J 5.7 Hz,
CH2O), 4.17 (2 H, s, ArCH2N), 7.32 (2 H, s, Ar), 7.48 (2 H, s, Ar); 13C NMR
(125 MHz, CD3OD): d=27.8, 30.6, 35.6, 45.8, 53.0, 71.8, 122.9, 128.0,
130.1, 131.1, 131.3, 140.0, 151.2, 156.6; HRMS-ESI m/z calcd. for
[C24H32Cl3NO2+H]+: 472.1576, found: 472.1573).
N-(3,5-Di-tert-butylbenzyl)-3-(2,6-dimethylphenoxy)propan-1-amine
(24). A mixture of 3-(2,6-dimethylphenoxy)propan-1-amine (316 mg, 1.76
mmol), 3,5-di-tert-butylbenzyl bromide (250 mg, 0.88 mmol) and dry THF
(25 mL) was heated at reflux for 24 h. The mixture was concentrated under
reduced pressure, 10% aq. NaOH (50 mL) was added and the mixture
extracted with EtOAc (2 ´ 50 mL). The combined organic extracts were
washed with sat. aq. NaCl, dried (MgSO4), filtered and concentrated under
reduced pressure. Purification by Protocol B (MeOH/H2O) afforded the title
compound (146 mg, 20%). 1H NMR (500 MHz, CD3OD): d=1.34 (18 H, s,
2 ´ C(CH3)3), 2.20-2.25 (8 H, m, 2 ´ CH3,CH2CH2N), 3.35-3.39 (2 H, m,
CH2N), 3.88 (2 H, t, J 5.8 Hz, CH2O), 4.27 (2 H, s, ArCH2N), 6.90 (1 H, t,
J 7.5 Hz, Ar), 7.00 (2 H, dd, J 7.4, 0.5 Hz, Ar), 7.39 (2 H, d, J 1.8 Hz, Ar),
7.55 (1 H, t, J 1.8 Hz, Ar); 13C NMR (125 MHz, CD3OD): d=16.4, 28.1, 31.8,
35.8, 46.5, 53.0, 70.0, 124.8, 125.2, 125.3, 130.0, 131.7, 131.8, 153.3,
156.6; HRMS-ESI m/z calcd. for [C26H39NO+H]+: 382.3109, found:
382.3107.
ii) 4-(3-Azidopropoxy)-1,3,5-trimethyl-1H-pyrazole (49). Prepared from
48, as per 28, except that the reaction was heated at 60 °C for 5 h, then
100 °C for 2 h. Flash chromatography (4:1 toluene/acetone) gave the title
compound (168 mg, 94%). 1H NMR (400 MHz, CDCl3): d=1.83-1.89 (2 H,
m, CH2CH2O), 2.06 (3 H, s, CH3), 2.08 (3 H, s, CH3), 3.44 (2 H, t, J 6.7 Hz,
CH2N), 3.55 (3 H, s, CH3), 3.76 (2 H, t, J 6.0 Hz, CH2O); 13C NMR (100
MHz, CDCl3): d=8.3, 10.6, 29.3, 36.1, 48.1, 71.1, 128.7, 138.1, 138.8;
HRMS-ESI m/z calcd. for [C9H17N3O+H]+: 210.1349, found: 210.1344).
iii)
3-(1,3,5-Trimethyl-1H-pyrazol-4-yloxy)propan-1-amine
(50).
Prepared from 49, as per 29, and purification by flash chromatography
(93:5:2 toluene/MeOH/Et3N) gave the title compound (110 mg, 70%). 1H
NMR (400 MHz, CDCl3): d=1.74 (2 H, quintet, J 6.6 Hz, CH2CH2O), 2.05
(3 H, s, CH3), 2.07 (3 H, s, CH3), 2.81 (2 H, t, J 6.9 Hz, CH2N), 3.54 (3 H,
s, CH3), 3.76 (2 H, t, J 6.2 Hz, CH2O); 13C NMR (100 MHz, CDCl3): d=8.4,
10.7, 33.5, 36.18, 36.20, 39.1, 72.8, 128.8, 138.2, 139.1; HRMS-ESI m/z
calcd. for [C9H19NO+H]+: 184.1444, found: 184.1454.
3-(2,6-Dimethylphenoxy)-N-(4-(trifluoromethoxy)benzyl)propan-1-
amine (25). Method A1 applied to 3-(2,6-dimethylphenoxy)propylamine
and 4-methoxylbenzaldehyde, followed by purification using Protocol A
(MeOH/H2O) afforded the title compound (157 mg, 72%). 1H NMR (500
MHz, CD3OD): d=2.22-2.26 (8 H, m, 2 ´ CH3,CH2CH2N), 3.37-3.40 (2 H,
m, CH2N), 3.88 (2 H, t, J 5.9 Hz, CH2O), 4.33 (2 H, s, ArCH2N), 6.90 (1 H,
t, J 7.5 Hz, Ar), 7.00 (2 H, d, J 7.4 Hz, Ar), 7.39-7.41 (2 H, m, Ar), 7.66-
7.68 (2 H, m, Ar); 13C NMR (125 MHz, CD3OD): d=16.4, 28.2, 46.6, 51.5,
iv)
2,6-Di-tert-butyl-4-((3-(1,3,5-trimethyl-1H-pyrazol-4-
yloxy)propylamino)methyl) phenol (22). Method A1 applied to 50 and
12
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10.1002/cmdc.201800781
ChemMedChem
FULL PAPER
69.9, 120.8 (1C, q, J 256.1 Hz, CF3), 122.7, 125.3, 130.0, 131.7, 131.7,
133.2, 151.3, 156.6; HRMS-ESI m/z calcd. for [C19H22F3NO2+H]+:
354.1675, found: 354.1675.
All experiments were conducted at ambient temperature. Eight
concentrations were applied to naïve cells (n = 4, where n = the number of
replicate wells/concentration) via steel needles of a multi-channel pipettor.
Vehicle control was applied for a 5 min exposure interval (n = 8). To verify
the sensitivity of the assay to block, lamotrigine and lidocaine were applied
for a 10 min exposure interval (n = 4). All test and control solutions
contained 0.3% DMSO and 0.05% Pluronic F-127. Each test article
formulation was sonicated (Model 2510/5510, Branson Ultrasonics,
Danbury, CT), at ambient room temperature for at least 20 min to facilitate
dissolution.
4-((3-(2,6-
Dimethylphenoxy)propylamino)methyl)benzenesulfonamide
(26).
Method A1 applied to 3-(2,6-dimethylphenoxy)propylamine and 4-
aminosulfonylbenzaldehyde, followed by purification using Protocol B
(MeOH/H2O) afforded the title compound (277 mg, 62%). 1H NMR (500
MHz, CD3OD): d=2.27-2.37 (8 H, m, 2 ´ CH3,CH2CH2N), 3.40 (2 H, t, J 8.0
Hz, CH2N), 3.89 (2 H, t, J 6.0 Hz, CH2O), 4.38 (2 H, s, ArCH2N), 6.90 (1 H,
t, J 7.5 Hz, Ar), 7.00 (2 H, d, J 7.5 Hz), 7.74 (2 H, d, J 8.0 Hz, Ar), 8.00 (2
H, d, J 8.0 Hz, Ar); 13C NMR (125 MHz, CD3OD): d=16.4, 28.2, 46.8, 51.7,
69.9, 125.3, 128.0, 130.0, 131.7, 136.6, 146.4, 156.6; HRMS-ESI m/z
calcd. for [C18H24N2O3S+H]+: 349.1585, found: 349.1588.
Data acquisition and analyses were performed using the IonWorks
QuattroTM or Barracuda system software (Molecular Devices Corporation,
Union City, CA) according to protocols 1 or 2. The decrease in current
amplitude after test article application was used to calculate the percent
block relative to control at the various test pulses as follows:
In vitro metabolic stability
Block(%) = (1 – ITP,TA / ITP,control) × 100%
(1)
The metabolic stability assay was performed by incubating each test
compound in liver microsomes at 37°C and a protein concentration of 0.4
mg/mL. The metabolic reaction was initiated by the addition of an NADPH-
regenerating system and quenched at various time points over a 60 min
incubation period by the addition of acetonitrile containing diazepam as
internal standard. Control samples (containing no NADPH) were included
(and quenched at 2, 30 and 60 minutes) to monitor for potential
degradation in the absence of cofactor. The human liver microsomes used
in this experiment were supplied by XenoTech, lot#1410230. Microsomal
incubations were performed at a substrate concentration of 1 μM.
Concentration-response data were fit to the following equation:
Block(%) = %VC + ((100 - %VC) / [1 + (C / IC50)N])
(2)
where C is the concentration of test article, IC50 is the concentration of the
test article producing half-maximal inhibition, N is the Hill coefficient, %VC
is the percentage of the current run-down (the mean current inhibition at
the vehicle control) and Block(%) is the percentage of ion channel current
inhibited at each concentration of a test article. Nonlinear least squares fits
were solved with the XLfit add-in for Excel (Microsoft, Redmond, WA).
Data analysis: Species scaling factors from Ring et al.[30] were used to
convert the in vitro CLint (μL/min/mg) to an in vivo CLint (mL/min/kg).
Hepatic blood clearance and the corresponding hepatic extraction ratio
(EH) were calculated using the well stirred model of hepatic extraction in
each species, according to the "in vitro T1/2" approach described in
Obach[31]. The EH was then used to classify compounds as low (<0.3),
intermediate (0.3-0.7), high (0.7-0.95) or very high (>0.95) extraction
compounds. Predicted in vivo clearance values have not been corrected
for microsomal or plasma protein binding. Species scaling calculations are
based on two assumptions: 1) NADPH-dependent oxidative metabolism
predominates over other metabolic routes (i.e. direct conjugative
metabolism, reduction, hydrolysis, etc.), and; 2) rates of metabolism and
enzyme activities in vitro are truly reflective of those that exist in vivo. If
significant non-NADPH-mediated degradation is observed in microsome
control samples, then assumption (1) is invalid and predicted clearance
parameters are therefore not reported.
6 Hz psychomotor seizure model of partial epilepsy
Tests were conducted by the Epilepsy Therapy Screening Program
(ETSP) through the National Institute of Neurological Disorders and Stroke
(NINDS). The test protocol was that reported in Barton et al.[24]
Acknowledgements
We are grateful to the National Institute of Neurological Disorders
and Stroke (NINDS) Epilepsy Therapy Screening Program
(ETSP) for in vivo mouse studies of the antiseizure properties of
compound 2. We thank FastForward (USA) for project funding,
and invaluable advice from Dr John Lowe.
Keywords: membrane proteins • ion channels • neurological
Voltage-gated Na+ channels
agents • anticonvulsant • propylamine
Sodium channel activity was studied under contract at ChanTest (Ohio,
USA) using a whole-cell patch clamp technique in CHO cells that were
stably transfected with human NaV1.2 (SCN2A) and NaV1.6 (SCN8A)
cDNAs were incorporated into an expression plasmid along with antibiotic
resistance genes. Cells were cultured in Ham's F-12 supplemented with
10% fetal bovine serum, 100 U/mL penicillin G sodium, 100 µg/mL
streptomycin sulfate and appropriate selection antibiotics. Before testing,
cells in culture dishes were washed twice with Hank’s Balanced Salt
Solution and treated with Accutase for approximately 20 min. Immediately
before use the cells were washed in HEPES-buffered physiological saline
to remove the Accutase and re-suspended in HEPES-buffered
physiological saline.
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Entry for the Table of Contents
New kids on the block: Achiral analogues of mexiletine were identified as selective blockers of human voltage gated sodium
channel 1.2. Compound 2 was highly potent for NaV1.2 and 500 times less potent in inhibiting NaV1.6 channels; compound 4 was the
most potent blocker but is non-selective against both channels. Compound 2 inhibited seizures induced by 6 Hz 44 mA electrical
stimulation in a kindled mouse model of refractory epilepsy.
15
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