Synthesis of a Series of Structurally Diverse MB327 Derivatives and Their Affinity Characterization at the Nicotinic Acetylcholine Receptor

Article abstract of DOI:10.1002/cmdc.201800325

A novel series of 30 symmetric bispyridinium and related N-heteroaromatic bisquaternary salts with a propane-1,3-diyl linker was synthesized and characterized for their binding affinity at the MB327 binding site of nicotinic acetylcholine receptor (nAChR) from Torpedo californica. Compounds targeting this binding site are of particular interest for research into new antidotes against organophosphate poisoning, as therapeutically active 4-tert-butyl-substituted bispyridinium salt MB327 was previously identified as a nAChR re-sensitizer. Efficient access to the target compounds was provided by newly developed methods enabling N-alkylation of sterically hindered or electronically deactivated heterocycles exhibiting a wide variety of functional groups. Determination of binding affinities toward the MB327 binding site at the nAChR, using a recently developed mass spectrometry (MS)-based Binding Assay, revealed that several compounds reached affinities similar to that of MB327 (pK_i=4.73±0.03). Notably, the newly prepared lipophilic 4-tert-butyl-3-phenyl-substituted bispyridinium salt PTM0022 (3 h) was found to have significantly higher binding affinity, with a pK_i value of 5.16±0.07, thus representing considerable progress toward the development of more potent nAChR re-sensitizers.

Full text of DOI:10.1002/cmdc.201800325

Accepted Article
Title: Synthesis of a Novel Series of Structurally Different MB327
Derivatives and Their Affinity Characterization at the Nicotinic
Acetylcholine Receptor
Authors: Klaus Theodor Wanner, Sebastian Rappenglück, Sonja
Sichler, Georg Höfner, Thomas Wein, Karin V. Niessen,
Thomas Seeger, Franz F. Paintner, Franz Worek, and Horst
Thiermann
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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.201800325
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800325
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ChemMedChem
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FULL PAPER
Synthesis of a Novel Series of Structurally Different MB327 Derivatives and
Their Affinity Characterization at the Nicotinic Acetylcholine Receptor
Sebastian Rappenglück[a]+, Sonja Sichler[a]+, Dr. Georg Höfner , Dr. Thomas Wein , Karin V.
[a]
[a]
[b]
[b]
[a]
[b]
Niessen , Dr. Thomas Seeger , Prof. Dr. Franz F. Paintner , Prof. Dr. Franz Worek , Prof. Dr. Horst
[b]
[a]
Thiermann , Prof. Dr. Klaus T. Wanner*
+
These authors contributed equally as first authors.
[
a]
b]
S. Rappenglück, S. Sichler, Dr. G. Höfner, Dr. T. Wein, Prof. Dr. F.F. Paintner, Prof. Dr. K.T. Wanner ORCID:0000-0003-4399-1425
Department of Pharmacy – Center for Drug Research
Ludwig-Maximilians-Universität München
Butenandtstr. 5-13, 81377 Munich, Germany
E-mail: : klaus.wanner@cup.uni-muenchen.de
[
K.V. Niessen, Dr. T. Seeger, Prof. Dr. F. Worek, Prof. Dr. H. Thiermann
Bundeswehr Institute of Pharmacology and Toxicology
Neuherbergstr. 11, 80937 Munich, Germany
Supporting information for this article is given via a link at the end of the document.
Abstract: A novel series of 30 symmetric bispyridinium and related
N-heteroaromatic bisquaternary salts with a propane-1,3-diyl linker
was synthesized and characterized for their binding affinity at the
MB327 binding site of nAChR (nicotinic acetylcholine receptor) from
Torpedo californica. Compounds targeting this binding site are of
particular interest for the research into new antidotes against
organophosphate poisoning, as therapeutically active 4-tert-butyl
substituted bispyridinium salt MB327 was previously identified as
nAChR resensitizer. Efficient access to the target compounds was
provided by newly developed methods enabling N-alkylation of
sterically hindered or electronically deactivated heterocycles
exhibiting a wide variety of functional groups. Determination of binding
affinities towards the MB327 binding site at the nAChR, using a
recently developed mass spectrometry (MS) based Binding Assay,
revealed that several compounds reached affinities similar to that of
overstimulation is precluded by their therapeutic limitations.[4]
Thus ongoing overstimulation by accumulated ACh forces the
[5,6]
nAChRs into
a
desensitized state,
which is currently
therapeutically inaccessible.[
7]
Resulting disturbance of
neuromuscular transmission may cause paralysis of respiratory
and other skeletal muscles as most severe and often lethal
consequences of OPC poisoning.
Current medical countermeasures make use of oximes such
as obidoxime or pralidoxime to reactivate the inhibited AChE, thus
treating cholinergic crisis. However, in many cases oximes were
shown to be ineffective. Fast dealkylation of the AChE-
organophosphonate complex in case of soman intoxication or
very slow reactivation rates of tabun-inhibited AChE for most
commonly used oximes are only two of several examples for
which the reactivation kinetic of this phenomenon has been well
[
8,9]
MB327 (pK
butyl-3-phenyl substituted bispyridinium salt PTM0022 (3h), was
found to have a significantly higher binding affinity with a pK value of
.16 ± 0.07, thus representing a considerable progress towards the
i
= 4.73 ± 0.03). Notably, newly prepared lipophilic 4-tert-
studied.
Hundreds of different oximes have been developed
and tested over the last decades, without the successful
10]
i
identification of
a
universal reactivator of AChE.[
In
5
consequence, restoration of nAChR-mediated cholinergic
signaling in the event of OPC poisoning remains a highly
challenging task demanding for an alternative approach not
exclusively based on AChE reactivation.
development of more potent nAChR resensitizers.
One very promising strategy might be the direct intervention
at the nAChR to recover its activity from desensitization as
Introduction
recently demonstrated in measurements using
a
novel
The recent use of sarin as chemical weapon in Syria as well
as the high number of fatal poisonings with organophosphorus
2
electrophysiological (SURFE R, surface electronic event reader)
[11]
platform by Niessen et al. These studies revealed that the 4-
pesticides
underline
the
threat
emanating
from
1-3]
tert-butyl substituted bispyridinium compound MB327 can
organophosphorus compounds (OPCs) to humanity.[
Therefore, there is an urgent need for effective medical treatment
of OPC intoxications. OPCs inactivate the enzyme
acetylcholinesterase (AChE), which results in accumulation of
acetylcholine (ACh) in the synaptic cleft of both muscarinic and
nicotinic synapses. Whereas overstimulation of muscarinic
receptors (mAChRs) can be antagonized by using atropine,
administration of anticholinergics to counteract nAChR
resensitize the desensitized nAChR and restore channel
[11,12]
function.
As previous investigations have already shown that
MB327 targets the orthosteric binding site with only weak affinity,
functional recovery of the nAChR is suggested to be mediated
[13,14]
rather by allosteric modulation.
In contrast to an oxime-based
therapy aiming at reactivation of inhibited AChE, an allosterically
mediated resensitization of the nAChR has the advantage to
restore nAChR activity and thereby, preserving neuromuscular
1
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ChemMedChem
10.1002/cmdc.201800325
FULL PAPER
function in any case of OP (organophosphorus) nerve agent
poisoning.
The therapeutic effect of MB327 against OPC poisoning has
also been demonstrated in several ex vivo, in vitro as well as in
vivo studies. In case of tabun-poisoned guinea pigs, for example,
treatment with MB327 significantly increased the survival rate as
structural changes affect affinity to the MB327 binding site should
provide information on the molecular interactions important for the
binding event. MB327 is a symmetric bispyridinium diiodide with
a tert-butyl group in 4-position of each pyridinium ring and a propyl
linker connecting both aromatic rings (Figure 1).
compared to the oxime HI-6, both administered together with
physostigmine and hyoscine.[15] Furthermore, Seeger et al. found
t-Bu
t-Bu
that
MB327
restores
soman-impaired
neuromuscular
N
N
transmission in human- and rat-respiratory muscle preparations
at concentrations of 100–200 µmol L . Unfortunately, MB327
–
1 [16]
2 I
also acts as an AChE inhibitor at slightly higher concentrations
MB327
–
1
(
IC50 about 600 µmol L ) and antagonizes nicotinic currents at
[
17,18]
concentrations ≥ 100 µ
M
19]
,
possibly leading to adverse effects,
Figure 1. Structure of MB327.
[
if administered in vivo. In consequence, the therapeutic window
of MB327 is too narrow for administration in case of OPC
poisoning, hence more potent and more selective nAChR
resensitizers have to be developed.
Depending on the structural characteristics, the influence of
which on the binding affinity should be studied, target compounds
were divided into four different types (I–IV), comprising in total 30
structurally related compounds (Figure 2). Compounds of type I
and II are bispyridinium salts substituted with either one lipophilic
Though MB327 has been studied extensively in the last years
regarding its potential use in treatment regimens,[20-22] the
targeted binding site at the nAChR was characterized only very
[
23]
recently. Binding Assays using mass spectrometry for marker
quantification (MS Binding Assays) enabling the affinity
determination for ligands targeting the MB327 binding site have
(
Me, i-Pr, t-Bu) or one hydrophilic (OMe, NMe2, N-
methylpyrrolidine) substituent on each pyridinium ring. These
moieties were introduced to investigate the influence of the
substituent position at the ring system, the steric demand of the
functional groups as well as hydrogen bridge interactions
between polar moieties and the binding pocket on the binding
affinity. Bispyridinium salts of type III were directly delineated from
the lead structure MB327 by introduction of an additional
substituent and designed to investigate the resulting affinity.
These compounds exhibit a 4-tert-butyl group as well as a second
functional group (e.g., Ph, CN, OMe, COOEt, Cl) in 2- or 3-
position of the pyridinium ring.
[
24]
been established.
could characterize the affinity of MB327 to the nAChR with a K
Based on these MS Binding Assays, we
i
–
1
value of 18.3 ± 2.6 µmol L , which is in line with its potency
determined in electrophysiological and pharmacological studies.
For our MS Binding Assays, we use the nAChR from Torpedo
californica, which shows a high degree of homology to the human
muscle-type nAChR and represents the most common model
[25,26]
system.
In parallel to the development of [ H
2
6
]MB327 MS Binding
Assays, our group identified two putative MB327 binding sites
using in silico docking studies at the nAChR of Torpedo
marmorata at 4°A resolution.[23] Respective sites are located
inside the channel, one in the extracellular and one in the
transmembrane domain. The developed MS Binding Assays
allow to gain quantitative binding data for the nAChR for the very
first time. The latter are an essential prerequisite for a detailed
understanding of the binding interactions at a molecular level and
for the development of valid structure-affinity relationships
representing a key element in the search for new antidotes
against OPC intoxication. Hence, we envisaged the synthesis and
affinity characterization of a novel series of structurally different
MB327 analogues, which were expected to be of interest to gain
first insights into structure-affinity relationships. Herein, we report
the results of this endeavor.
In consequence, from these compounds, substituent effects
can be directly derived by comparing the binding affinity with that
of MB327, which is devoid of any substituent in 2- or 3-position.
To investigate the importance of π-π interactions with aromatic
amino acid side chains of the nAChR during the binding event and
to expand the structural diversity amongst the test compounds,
pyridine was finally replaced by other heteroaromatic compounds
such as isoquinoline, thiazole or 2-tert-butylpyrazine, thus
resulting in new target structures of type IV.
Common to all newly synthesized compounds, however, is
the propane-1,3-diyl (C3) linker, known from MB327. This linker
was used for all test compounds, as MB327 showed
a
2
significantly higher resensitization potency in SURFE R-based
electrophysiological experiments as compared to structurally
analogous compounds with a shorter i.e. an ethane-1,2-diyl (C2)
[
11]
or a longer i.e. a butane-1,4-diyl (C4) linker.
Results and Discussion
Structure of Target Compounds
With MB327 as lead structure, we focused on the
investigation of symmetrical bispyridinium compounds with
distinct structural modifications. The results of how these
2
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ChemMedChem
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FULL PAPER
I
II
5
2
4
3
6
4
3
2
X
2 X
Ralkyl
Ralkyl
Rpolar
Rpolar
N
N
N
N
2
1a–h
2a–e
a
b
c
d
a
b
c
d
2-t-Bu
3-t-Bu
2-i-Pr
3-i-Pr
2-OMe
3-OMe
4-OMe
3-NMe2
e
f
g
h
e
4-i-Pr
3,5-Me2 2,6-Me2 2,4,6-Me3
3-N-methylpyrrolidine
III
IV
t-Bu
t-Bu
2
X
2 X
R
R
R
R
N
N
N
N
3
2
3a–l
4a–e
a
b
c
d
a
b
3-F
3-Cl
3-Br
3-CN
isoquinoline
2-tert-butylpyrazine
e
f
g
h
c
d
3-OMe 3-CONMe2 3-Me
3-Ph
1,3-thiazole
3-N-methylimidazole
i
j
k
l
e
3-C CH 3-COOEt
2-Me 3-COOH
N-tert-butylimidazole
–
–
–
Figure 2. Structures of envisaged target compounds (type I–IV). X = Cl , I , OTf
Chemistry
the intermediate isopropyl cuprate and by a β-H elimination of the
[
31]
putative (t-Bu)
out to be far less favorable for the substitution with the tert-butyl
cuprate, affording 8 in 12% only (43% in Et O). This is likely due
to the more reactive cuprate oligomers, present in Et O, as
2
Cu(III)pyridyl complex. Interestingly, THF turned
As shown in Figure 2, all target compounds are symmetric
bispyridinium salts or related from N-aromatic subunits derived
salts with a propane-1,3-diyl linker. As such, these compounds
are easily accessible in a two-step reaction sequence. In the first
step, the required nitrogen heterocycles are synthesized which in
the second step afford the target compounds upon N-alkylation.
Of the 30 nitrogen heterocycles employed in the synthesis of the
target compounds, 25 were either commercially available or
synthesized according to literature procedures,[27-29] including the
2
2
compared to the less reactive monomers, predominant in THF,
which is a specific feature of cyano cuprates found by Krause and
[
32]
Gschwind. Besides, yields were also negatively affected by the
remarkable high volatility of 6 and 8.
3
-substituted 4-tert-butylpyridine derivatives, the preparation of
a
[
30]
which was recently reported by us. The remaining five pyridine
derivatives namely, 4-(tert-butyl)-2-methylpyridine (15), 3-tert-
butylpyridine (8), 2- (6), 3- (11), and 4-isopropylpyridine (13) were
prepared as described below.
N
Br
Br
N
i-Pr
5
6
Preparation of alkyl substituted pyridine derivatives
t-Bu
b
2-Isopropylpyridine (6) as well as 3-tert-butylpyridine (8) were
prepared from the corresponding bromopyridines 5 and 7 by
cuprate mediated substitution reactions resulting in low to
moderate yields of 19 and 43%, respectively, for the target
compounds (Scheme 1).[27] The low yields are to be assigned to
side reactions that led to pyridine as side product, which in each
of the two cases was isolated in large amounts. These side
reactions might result from a halogen metal exchange caused by
N
N
7
8
Scheme 1. Synthesis of alkyl pyridines 6 and 8. Reagents and conditions: a)
CuCN (4.0 eq.), i-PrMgCl (8.0 eq.), –78 °C for 2 h → RT for 15 h, THF, 19%; b)
CuCN (4.0 eq.), t-BuMgCl (8.0 eq.), –78 °C for 2 h → RT for 15 h, Et
2
O, 43%
(
12% yield in THF).
3
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As 3-isopropylpyridine (11) was found to be not accessible by
a cuprate mediated substitution of 7, it was synthesized in two
steps from tertiary alcohol 9.[33] Acid catalyzed dehydration of 9
and subsequent hydrogenation of the resulting alkene 10 with
equivalents of the pyridine derivative 8, devoid of any substituents
in 2- and 6-position and of any electron withdrawing groups,
required 72 h at 70 °C in DMF for full conversion, affording 1b in
79% yield (Scheme 4).
2
Pd/C and H afforded 11 in 51% yield (Scheme 2).
t-Bu
a
2
I
Me
OH
N
N
i-Pr
t-Bu
t-Bu
N
a
Me
b
I
I
3
Me
8
1b
1
6
N
N
N
9
10
11
Scheme 4. Synthesis of 1b. Reagents and conditions: 1,3-diiodopropane (16,
1.0 eq.), 8 (2.5 eq.), DMF, 70 °C, 72 h, 79%.
Scheme 2. Synthesis of 11. Reagents and conditions: a) conc. AcOH / conc.
SO (1:2.7), 150 °C, 30 min; b) Pd/C (5 mol%), H , MeOH, 16 h, 51% over
both steps.
H
2
4
2
Hence, 1,3-diiodopropane (Scheme 4) did not seem suitable
for an efficient preparation of the desired target compounds. This
problem could, however, be overcome employing the far more
reactive propane-1,3-diyl bis(trifluormethanesulfonate) (21). With
bistriflate 21, compounds of type I–III (1a, c–h, Table 1; 2a–e,
Table 2; 3a–l, Table 3) were easily accessible in fair to excellent
yields (47–99%) by alkylation of the corresponding heterocycles.
The synthesis of 4-isopropylpyridine (13) and 4-(tert-butyl)-2-
methylpyridine (15) was accomplished in a two-step reaction
starting from 12 and 14, respectively (scheme 3). Treatment of 12
with AcCl and of 14 with PhOCOCl followed by an addition of the
respective organocuprates to the thus formed N-acyliminium
intermediates provided after oxidation 13 and 15 in 33 and 47%
yield, respectively.
2 2
Reactions were performed either at 50 °C in CH Cl or, if
necessary, at higher temperatures without a solvent. Resulting
bis(trifluoromethanesulfonate) salts were finally purified by
crystallization without any problems.
i-Pr
Table 1. Synthesis of bispyridinium compounds of type I (1a, c–h).
a, b
Ralkyl
Ralkyl
Ralkyl
N
N
a
12
13
N
N
N
TfO 3OTf
t-Bu
2
TfO
6
, 11, 13
17–20
21
c, d
1a, c–h
Entry
Starting Material
T
t
Product
Yield
(%)
N
Me
N
Me
14
15
R
alkyl
No.
(°C)
(min)
1
Scheme 3. Synthesis of 13 and 15. Reagents and conditions: a) AcCl (1.0 eq.),
THF, –78 °C, 75 min, then (i-Pr) CuCN(MgCl) (1.1 eq.) 80 min; b) DDQ (1.0
eq.), CH Cl , RT, 70 min, 33% over both steps; c) PhOCOCl (1.0 eq.), t-BuZnBr
1.1 eq.), CuCN (1.1 eq.), LiCl (2.2 eq.), –78 °C for 5 h → RT for 16 h, THF; d)
1
2
3
4
5
6
7
2-t-Bu
2-i-Pr
17
6
85
50
50
50
50
70
50
300
5
a
c
d
e
f
73
77
88
81
69
75
81
2
2
2
2
(
3-i-Pr
11
13
18
19
20
2
S
8
(1.1 eq.), naphthalene (15 eq.), 200 °C, 20 min, mw: 300 W, 47% over both
steps.
4-i-Pr
60
1
3,5-Me
2
2
Preparation of the target compounds
2,6-Me
1
g
h
As mentioned above, the synthesis of symmetric
bis(pyridinium) salts with a propane-1,3-diyl linker (Figure 2) is
commonly accomplished by reaction of 1-3-diiodo- or 1,3-
dibromopropane with an excess of the respective pyridine
derivatives in DMF or MeCN at 70 °C.[34-36] However, when these
reaction conditions were used for the synthesis of compounds of
type I and II, in most cases, only a negligible conversion but
substantive decomposition of reactants occurred. This is mainly
attributed to the low reactivity of the alkyl halides, especially when
sterically hindered 2-substituted pyridine derivatives were
employed (6, 17, 19, 20). Besides, in case of N-alkylated 22 and
2,4,6-Me
3
15
Reagents and conditions: a) pyridine derivatives (2.5 eq.), 21 (1.0 eq.), no
solvent, T, t.
In contrast to the above-mentioned alkylation reaction with
,3-diiodopropane (16), the high reactivity of bistriflate 21 allowed
short reaction times (1–30 min in most cases) and gave also
access to bispyridinium compounds with sterically demanding
substituents in 2-position such as 1a, 1c, 1g, 1h (Table 1), and 3k
1
(
Table 3).
23, also demethylation reactions by iodide ions occurred. Notably,
even for the reaction of 1,3-diiodopropane (16) with 2.5
4
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ChemMedChem
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FULL PAPER
terminal alkyne (3i), ester (3j), amide (3f), and even secondary
Table 2. Synthesis of bispyridinium compounds of type II (2a–e).
(
2d) and tertiary amino substituents (2e) were well tolerated. Also,
Rpolar
Rpolar
Rpolar
the carboxylic acid 3l (Table 3, entry 12) could be synthesized by
basic hydrolysis of the ester function of 3j with aqueous NaOH.
a
To this end, however, milder conditions had to be employed
N
N
N
–1
(
hydroxide ion concentration of only 0.055ꢀmol L ), to reduce a
2
TfO
Zincke-König type cleavage to a minimum, thus affording 3l in
2
2–25,
66% yield after crystallization.
rac–26
2a–e
Although biological evaluation of synthesized bispyridinium
Entry
Starting Material
t
Product
Yield
(%)
salts 1a–h (Table 1), 2a–e (Table 2) and 3a–l (Table 3) using our
MS Binding Assays yielded reliable results, the triflate counterion
was assumed to cause interferences in electrophysiological
measurements on the SURFE R platform. Thus, we decided to
synthesize compounds of type IV with iodide instead of triflate
counterions.
In consequence, compounds 4a–e were synthesized by
reaction of the respective N-heteroaromatics 37–41 with 1,3-
diiodopropane (16) in MeCN under microwave heating to 90–
R
polar
No.
(min)
2
2
[37]
1
2
3
4
5
2-OMe
3-OMe
4-OMe
22
23
1
1
a
b
c
d
e
67
83
84
47
72
24
1
3-NMe
2
25
10
30
120 °C. The yields were good to excellent (78–92%, Table 4).
3-N-methylpyrrolidine
rac-26
Under microwave conditions, higher reaction temperatures could
be reached resulting in distinctly reduced reaction times. Besides,
also side reactions appeared to be absent this time.
Reagents and conditions: a) pyridine derivatives (2.5 eq.), 21 (1.0 eq.), 50 °C, t,
entry 1-4: no solvent, entry 5: reaction in CH Cl
2
2
.
Solubility of compounds of type I–IV in water proved to be
sufficient for biological testing and allowed the preparation of
solutions with concentrations of at least 2.5 mM. Interestingly, a
Table 3. Synthesis of bispyridinium compounds of type III (3a–l).
faster dissolution in water was observed for triflate salts of type I–
III compared to iodide salts of type IV.
t-Bu
t-Bu
t-Bu
a
R
R
R
N
N
N
Table 4. Synthesis of non-bispyridinium compounds of type IV (4a–e).
2
TfO
a
3a–k, l[a]
2 I
2
7–36, 15
R
R
R
N
N
N
Entry
Starting Material
t
Product
Yield
(%)
3
7–41
4a–e
R
No.
(min)
3
Entry
Starting Material
T
t
Product
Yield
1
2
3
4
5
6
7
8
9
3-F
3-Cl
27[b]
28
29
30
31
32
33
34
35
36
15
3j
4
30
30
40
5
a
b
c
d
e
f
85
81
99
85
83
77
89
99
91
98
82
66
N
het.
No.
(°C)
90
(h)
4
(%)
3-Br
1
2
3
4
5
37
38
39
40
41
3
16
16
1
a
b
c
d
e
86
3-CN
3-OMe
3-CONMe
3-Me
120
120
90
78
87
92
82
2
30
20
30
40
30
30
16 h
g
h
i
3-Ph
3-C≡CH
3-COOEt
2-Me
N
1
0
1
j
Me
N
1
k
l
1
2[a]
3-COOEt
90
1
Reagents and conditions: a) pyridine derivatives (2.5 eq.), 21 (1.0 eq.), CH
0 °C, t. [a] 3l was synthesized from 3j (1.0 eq.) in 66% yield by basic hydrolysis
using aq. NaOH (0.1 , 2.5 eq.) in H O/MeCN (5:1) at RT for 16 h. [b] 4-(tert-
2 2
Cl ,
5
Reagents and conditions: a) Nhet. (2.5 eq.), 16 (1.0 eq.), MeCN, T, t, mw: 150W.
M
2
butyl)-3-fluoropyridine was released in situ from the corresponding hydro
chloride 27.
To evaluate the stability of synthesized target compounds in water,
NMR measurements of a random selection of compounds (1b, 2a,
Despite the high reactivity of bistriflate 21, a variety of functional
groups, i.e. alkylarylethers (2a–c, 3e), nitrile (3d), halide (3a–c),
2c, 3d, 3l) after 12 h incubation in D O at room temperature were
performed and provided no indications for hydrolysis.
2
5
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ChemMedChem
10.1002/cmdc.201800325
FULL PAPER
Preparation of deuterated MB327 analogs for MS Binding
Assays
provided the corresponding bistriflate 45 in 58% yield over both
steps. Reaction of 45 with 2.5 equivalents 4-tert-butylpyridine
finally afforded 46 in a yield of 77%.
The synthesis of [ H18]MB327 (49) started from pyridine (12)
and was performed in three steps, the total yield amounting to
For the recently established MS Binding Assays targeting the
2
MB327 binding site at the Torpedo-nAChR, deuterated MB327
2
2
6
analogs [ H ]MB327 (46) and [ H18]MB327 (49) were required as
25% and the deuteration degree to ≥ 99% (Scheme 6). In the first
MS marker and internal standard, respectively. Their synthesis
was accomplished as follows.
The deuterium labeled compound 46 was synthesized in four
steps starting from malonic ester 42 in an overall yield of 18% and
a deuteration degree of ≥ 99% (Scheme 5). At first, 42 was treated
step, pyridine (12) was treated with AcCl to generate the
corresponding N-acylpyridinium ion in situ which upon trapping
with (t-Bu)
79:21 for C4-isomer). Oxidation of 1,4-dihydropyridine 47 with
DDQ led to d -labeled pyridine 48 in 31% yield which upon
reaction of triflate 21 with 2.5 equivalents provided 49 in 81% yield.
2 2
CuCN(MgCl) afforded 47 with good regioselectivity
(
9
with D
2
O and pyridine according to Cocker et al. affording the d
in Et O and
2
-propanediol 44 with Tf O
2
-
derivative 43 in 40% yield.[38] Reduction with LiAlD
4
2
subsequent treatment of crude d
6
2
Scheme 5. Synthesis of [ H
6 2 4 2
]MB327 (46). Reagents and conditions: a) pyridine (6.0 eq.), D O (12 eq.), RT, 20 h, 40%, 99.6% D; b) LiAlD (3.0 eq.), Et O, 50 °C,
5
.5 h; c) Tf
2
O (2.0 eq.), pyridine (2.0 eq.), CH
2
Cl
2
, –78 °C → RT, 3 h, 58% over both steps; d) 4-tert-butylpyridine (2.5 eq.), CH Cl , 50 °C, 30 min, 77%, 99% D.
2
2
2
Scheme 6. Synthesis of [ H18]MB327 (49). Reagents and conditions: a) AcCl (1.0 eq.), THF, –78 °C, 60 min, then d
9
-t-BuMgCl (2.2 eq.), CuCN (1.1 eq.), THF, –
2 2
78 °C, 1.5 h; b) DDQ (1.0 eq.), CH Cl , RT, 1 h, 31% over both steps; c) 21 (0.4 eq.), 50 °C, 2.5 h, 81%, 99% D.
–1
and 1.95 mmol L–
Biological evaluation
NaCl, 5 mmol L KCl, 8.05 mmol L–1 Na
2
HPO
4
1
2 4
NaH PO , pH 7.4) were thawed, directly diluted with incubation
All target compounds synthesized in the context of this study
were catalogued with a certain PTM number (Pharmacy and
Toxicology Munich). Binding affinities towards the MB327 binding
site at the Torpedo-nAChR were determined in MS Binding
buffer and incubated in presence of the marker (and test
compounds if required). Both buffers, storage and incubation
buffer, are almost identical in this assay, except that the latter
–
1
2
additionally contains 1 mmol L CaCl . With the aim to simplify
2
[24]
6
Assays using [ H ]MB327 (46) as marker (Table 5).
the developed MS Binding Assays, we examined the possibility to
use only one buffer for the whole procedure, i.e. for the storage of
Torpedo membrane preparations as well as for the incubation in
the binding experiments described here. Since the storage buffer,
differing from the incubation buffer by the lack of CaCl , was also
2
identical to the incubation buffer that had been used in
3H]epibatidine binding studies,
With these MS Binding Assays, developed recently in our
group, it was possible for the first time to characterize binding of
MB327 towards the nAChR (pK
autocompetition experiments with [ H
According to the corresponding experimental protocol, aliquots of
i
of 4.75 ± 0.07 for MB327 in
2
6
]MB327 (46) as marker).
nAChR-enriched membranes, prepared from Torpedo californica
electroplaque tissue and frozen in storage buffer (120 mmol L
[18,37]
[
which were conducted
–1
recently for characterization of bispyridinium compounds at the
6
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ChemMedChem
10.1002/cmdc.201800325
FULL PAPER
orthosteric binding site of the Torpedo-nAChR, it appeared
substituted compounds 1a (PTM0002), 1b (PTM0001) and
2
obvious to choose this buffer also for incubation in [ H
6
]MB327
MB327 (pK
contrast, the 3-dimethylamino substituted bispyridinium salt 2d
(PTM0007) showed a higher pK value (4.78 ± 0.05, Table 5, entry
13) than the 3-tert-butyl substituted compound 1b (PTM0001, pK
= 4.44 ± 0.10, Table 5, entry 3). In conclusion, the NMe -group is
suggested as potential substitute for the tert-butyl-substituent.
The binding affinities of type III compounds exhibiting a F, Cl,
Br, CN, OMe, CONMe , Me, C≡CH or COOEt substituent in 3-
i
= 4.43 ± 0.12 to 4.73 ± 0.03, Table 5, entries 1–3). In
MS Binding Assays. Hence, saturation and autocompetition
experiments were conducted exactly as previously described,[24]
but employing the storage buffer also for incubation of binding
samples. Under these marginally modified conditions, we found a
i
i
2
–
1
K
d
of 14.2 ± 1.4 µmol L and a Bmax of 263 ± 30 pmol [mg protein]
–
1
2
(
n = 3) in saturation experiments using [ H
of 4.73 ± 0.03 (K
for autocompetition experiments using [ H
6
]MB327 (46) as
= 18.7 ± 1.2 µmol L , n = 3)
–1
marker and a pK
i
i
2
2
6
]MB327 (46) as marker
position (Table 5, entries 15–21 and 23–25) in addition to the 4-
tert butyl group in MB327 varied from 4.00 ± 0.12 in case of 3-CN
derivative 3d (PTM0019, Table 5, entry 18) to 4.80 ± 0.08 for 3-
alkyne substituted 3i (PTM0025, Table 5, entry 23) without a clear
tendency when regarding the nature of the substituent.
and native MB327 as competitor (for further details see
supplementary information). As there was no significant
difference (verified by two-sided t-test) between the affinity
constants, obtained in experiments with CaCl
experiments without CaCl
in the incubation buffer,[24] we decided
to use the CaCl free storage buffer also as incubation buffer in
2
as opposed to
2
However, the results indicate that the 4-tert-butyl group in the
bispyridinium salts has a beneficial effect on binding affinity. This
2
all binding experiments.
becomes evident by comparing the pK
substituted MB327 (pK = 4.73 ± 0.03, Table 5, entry 1) with those
of 3-methoxy substituted 2b (PTM0009, pK = 3.79 ± 0.09, Table
5, entry 11) and 4-tert-butyl-3-methoxy derivative 3e (PTM0016,
pK = 4.46 ± 0.07). Apparently, the addition of a tert-butyl group in
4-position increased the binding affinity of the 3-methoxy
derivative 2b (PTM0009) by 0.67 log units from 3.79 ± 0.09 to 4.46
± 0.07 for 3e (PTM0016, compare Table 5, entries 11 and 19).
In accordance with the negative effect of polar substituents on
binding affinity as discussed above, zwitterionic carboxylic acid 3l
(PTM0028) exhibited the lowest binding affinity of all tested
i
value of 4-tert-butyl
Although this marginal modification of the sample matrix was
not expected to affect the quantification of the MS marker, we still
decided to again examine the parameters selectivity, linearity,
lower limit of quantification (LLOQ), accuracy as well as precision
of the LC-ESI-MS/MS quantification method according to the
FDA’s guidance on bioanalytical method validation.[39] In short, all
requirements of the Center for Drug Evaluation and Research
i
i
i
(
CDER) guideline of the FDA regarding the above mentioned
parameters were fulfilled (for further detail see supplementary
information), clearly demonstrating LC-ESI-MS/MS quantification
to be robust and reliable also under these binding conditions. This
i
compounds with a pK value of 2.74 ± 0.13 (Table 5, entry 26).
The highest binding affinity, on the other hand, displayed the
2
modified protocol for [ H
6
]MB327 MS Binding Assays (for more
detail see experimental part) was finally used for all further
determinations of binding affinities of target compounds.
Initially, the influence of the position of the tert-butyl
substituent on the binding affinity was investigated by comparing
lipophilic 3-phenyl substituted MB327 analogue 3h (PTM0022)
with a pK
half a log unit higher than the pK
i
value of 5.16 ± 0.07 (Table 5, entry 22), which is about
value of MB327 (Table 5, entry
i
1) and two and a half order of magnitude above the one found for
carboxylic acid 3l (PTM0028, Table 5, entry 26). The statistical
significance of the higher affinity found for the phenyl substituted
the pK
i
value of 2-tert-butyl and 3-tert-butyl substituted
compounds 1a (PTM0002) and b (PTM0001) (type I) with that of
4-tert-butyl substituted MB327 (Table 5, entries 1–3). Interestingly, derivative 3h (PTM0022) as compared to MB327 was finally also
the difference between both 2- and 3-regioisomers was negligible,
but binding affinity decreased when the tert-butyl group was
moved from the 4- to the 3- or 2-position. Moreover, reduction of
the steric demand of the lipophilic substituent by replacing the tert-
butyl group in MB327, 1a (PTM0002) and 1b (PTM0001) by an
isopropyl group (1c–e, PTM0003, PTM0013, PTM0014, entries
verified by means of two-sided t-test (5% significance level). The
two additional aromatic phenylpyridinium rings present in 3h
(PTM0022) are likely to be involved in lipophilic interactions with
the target giving rise to an increase in affinity.
i
According to the pK values found for the non-bispyridinium
compounds of type IV (4a–e, PTM0032–PTM0034, PTM0057,
PTM0059, entries 27–31) none of the heterocyclic systems
studied revealed a significantly higher binding affinity than MB327.
Interestingly, electron poor 2-tert-butylpyrazinium salt 4b
4–6) led to a distinct reduction of binding affinity, except for the 3-
substituted compounds, where the difference was less
pronounced. A decrease in binding affinity was also observed for
the lutidine (1f, 1g, PTM0005 and PTM0004, entries 7 and 8) and
collidine derived bispyridinium salts (1h, PTM0006, entry 9),
especially when the 4-position remained unsubstituted (1f,
(PTM0057, pK
lower pK value as compared to the structurally related but far less
electron-deficient 3-tert-butylpyridinium salt 1b (PTM0001, pK
i
= 3.56 ± 0.09, Table 5, entry 28) showed a clearly
i
i
=
PTM0005: 3,5-Me2, pK
3.80 ± 0.04; 1h, PTM0006: 2,4,6-Me
compared to MB327: pK = 4.73 ± 0.03; see Table 5 entries 1 and
–9).
i
= 3.76 ± 0.14; 1g, PTM0004: 2,6-Me
2
, pK
i
4.44 ± 0.10, Table 5, entry 3). Binding affinity of bisimidazolium
salts decreased significantly upon transition from N-tert-butyl
=
3
, pK = 4.30 ± 0.09 as
i
i
substituted 4e (PTM0059, pK
N-methyl substituted 4d (PTM0034, pK
i
= 4.34 ± 0.13, Table 5, entry 31) to
= 3.55 ± 0.11, Table 5,
7
i
Comparing the affinities of the bispyridinium salts substituted with
lipophilic (type I, Table 5, entries 1–9) with those substituted with
polar residues of type II (Table 5, entries 10–14), it becomes
obvious that the replacement of the alkyl substituents by an N-
methylpyrrolidine moiety in 3-position (2e, PTM0027) or a polar
methoxy group (2a–c, PTM0008–PTM0010) in 2-, 3- or 4-position
entry 30). This indicates a general, beneficial effect of the tert-
butyl group over smaller groups that had similarly been observed
for the bispyridinium salts (compare e.g. MB327 with 1e
(PTM0013), Table 5, entries 1 and 6). Though being unsubstituted
and hence smaller than the N-methylimidazole derivative 4e
(PTM0059, Table 5, entry 31), the 1,3-thiazole derived compound
has a negative influence on binding affinity. The determined pK
i
i
4c (PTM0033) still had a reasonable affinity (pK 4.07 ± 0.13,
values range from 3.87 ± 0.13 to 3.97 ± 0.04 and are thus at least
Table 5, entry 29).
about half a log unit lower than those found for the tert-butyl
7
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ChemMedChem
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FULL PAPER
Table 5: Structures of synthesized target substances and binding affinities to the MB327 binding site.
Type I: 1a–h
Type II: 2a–e
5
4
3
6
4
3
2
TfO
2 TfO
Ralkyl
Ralkyl
Rpolar
Rpolar
N
N
N
N
2
2
Type III: 3a–l
TfO
Type IV: 4a–e
t-Bu
t-Bu
2
2 I
R
R
R
R
N
N
N
N
3
2
pK[
a]
PTM-Code
Entry
Compound
Ralkyl / Rpolar / R / Nhet.
i
1
2
3
4
5
6
7
8
9
MB327
1a
4-t-Bu
2-t-Bu
3-t-Bu
2-i-Pr
4.73 ± 0.03
4.43 ± 0.12
4.44 ± 0.10
3.83 ± 0.11*
4.18 ± 0.11
4.27 ± 0.11
3.76 ± 0.14*
3.80 ± 0.04*
4.30 ± 0.09
0002
0001
0003
0014
0013
0005
0004
0006
1b
1c
1d
3-i-Pr
1e
4-i-Pr
1f
3,5-Me
2
2
1g
2,6-Me
1h
2,4,6-Me
3
10
11
12
13
14
2a
2b
2c
2d
2e
2-OMe
3-OMe
4-OMe
3.87 ± 0.13*
3.79 ± 0.09*
3.97 ± 0.04*
4.78 ± 0.05
3.63 ± 0.13*
0010
0009
0008
0007
0027
3-NMe
2
3-N-methylpyrrolidine
15
16
17
18
19
20
21
22
23
24
25
26
3a
3b
3c
3d
3e
3f
3-F
3-Cl
4.23 ± 0.05*
4.54 ± 0.09
4.62 ± 0.08
4.00 ± 0.12*
4.46 ± 0.07
4.43 ± 0.10
4.57 ± 0.08
5.16 ± 0.07*
4.80 ± 0.08
4.50 ± 0.04
4.31 ± 0.09
2.74 ± 0.13*
0021
0020
0018
0019
0016
0024
0017
0022
0025
0015
0023
0028
3-Br
3-CN
3-OMe
3-CONMe
2
3g
3h
3i
3-Me
3-Ph
3-C≡CH
3-COOEt
2-Me
3j
3k
3l
3-COOH
27
28
29
30
31
4a
4b
4c
4d
4e
isoquinoline
2-tert-butylpyrazine
1,3-thiazole
4.77 ± 0.06
3.56 ± 0.09*
4.07 ± 0.13
3.55 ± 0.11*
4.34 ± 0.13
0032
0057
0033
0034
0059
N-methylimidazole
N-tert-butylimidazole
i
[a] Data are given as mean ± SEM of three independent experiments. pK values with a statistically significant difference towards MB327’s binding affinity are marked
by an asterisk (verified by means of two-sided t-test employing a 5% significance level).
8
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Notably, bis(isoquinolinium) salt 4a (PTM0032, pK
i
= 4.77 ±
2
affinities for the less polar derivatives. Interestingly, NMe was
0
.06), which is able to undergo more extensive lipophilic
identified to be the only but notable exception, revealing better
results than most apolar substituted compounds. In consequence,
the role of hydrogen bridge acceptors on the binding affinity is
worth to be investigated in more detail.
Finally, there is compelling evidence, that anionic functional
groups have a distinct negative influence on the binding process,
since the addition of two carboxylic acid functions to the structure
of MB327 resulted in a severe drop in binding affinity (3l,
PTM0028). To get deeper insights into the interaction of
bisquaternary salts with the MB327 binding site at the nAChR,
further studies based on structurally more diverse non-symmetric
bispyridinium compounds are under way.
interactions, was found to have the highest binding affinity of all
non-bispyridinium compounds, being in the same range as the
binding affinity of MB327. Altogether, the substitution of
bispyridinium compounds by addition of polar but uncharged
functional groups generally led to a slight decrease in binding
affinity, whereas the addition of a carboxylic acid function reduced
binding affinity dramatically (3l, PTM0028, Table 5, entry 26). The
sole exception is the 3-dimethylamino substituted bispyridinium
compound 2d (PTM0007, Table 5, entry 13), which shows a
slightly increased binding affinity as compared to MB327 (Table
5
, entry 1).
Notably, more lipophilic 3-phenyl-4-tert-butyl substituted
bispyridinium salt 3h (PTM0022, pK = 5.16 ± 0.07, Table 5, entry
2) revealed the highest binding affinity of all bispyridinium salts
i
2
Experimental Section
tested so far, representing an important progress in development
of new nAChR resensitizers with improved potency although its
pharmacological effects remain to be determined. Very recently,
Wein et al. described the putative binding site as composed of two
subdomains, one of which is lined with more hydrophilic and the
other with more lipophilic amino acids.[23] Accordingly, the design
of non-symmetric compounds displaying two different polar
termini, one with higher the other with lower polarity, could be a
promising strategy for increasing the binding affinity.
General Methods
Anhydrous reactions were carried out in vacuum-dried glassware under
argon atmosphere. Microwave reactions were performed in sealed glass
vials using a CEM Discover SP microwave synthesizer. THF, 1,4-dioxane
2 2
and CH Cl were distilled prior to use under nitrogen atmosphere and dried
[
40]
according to standard procedures. Et
2
O (technical grade) was purified
by distillation and dried by refluxing for 6 h over sodium without
benzophenone under nitrogen atmosphere before being distilled once
again prior to use. All other chemicals were used as purchased from
commercial sources and solvents were distilled before use. TLC was
carried out using plates purchased from Merck (silica gel 60ꢀF254 on
aluminum sheet). Flash chromatography (FC) was performed using Merck
silica gel 60 (40–63ꢀµm mesh size) as stationary phase. Melting points
were determined with a BÜCHI 510 melting point apparatus and are
uncorrected. IR spectroscopy was performed using an FT-IR
Spectrometer 1600 and Paragon 1000 (PerkinElmer); oils were measured
as film and solid samples as KBr pellets. High-resolution (HR) mass
spectrometry was performed on a Finnigan MAT 95 (EI), Finnigan LTQ FT
Conclusions
In summary, a set of 30 symmetric bispyridinium and related
salts with
a propane-1,3-diyl linker were synthesized and
characterized for their binding affinity towards the MB327 binding
site at the Torpedo-nAChR. Binding affinities were determined
using our recently developed MS Binding Assays and discussed
regarding MB327’s binding affinity, the most potent nAChR
resensitizer so far, as reference. To the best of our knowledge,
this represents the first study providing quantitative binding data
for the MB327 binding site, which, in turn, should be useful for
further developments in structure and ligand-based design of
nAChR resensitizers.
1
13
(
ESI) and Jeol JMS 700 MStation (FAB). H and C NMR spectra were
recorded with a Bruker BioSpin Avance III HD 400 and ꢀ500 MHz (equipped
with a Prodigy™ cryo probe) using TMS as internal standard and
integrated with MestReNova (Version 10.0.2), Mestrelab Research S.L.
2015. The purity of the test compounds was determined by means of
®
quantitative NMR using Sigma Aldrich TraceCERT maleic acid or
dimethylsulfone as internal calibrants.[
41,42]
The purity of all tested
All target compounds were synthesized via N-alkylation of the
respective aromatic N-heterocycles using either propane-1,3-
ditriflate or propane-1,3-diiodide as alkylating agents. Optimized
reaction conditions allowed very fast and clean reactions in the
presence of a wide variety of functional groups. Being also well
suited for the alkylation of less reactive nitrogen heterocycles,
such as sterically hindered 2-tert-butylpyridine or electron
deficient 2-tert-butylpyrazine, makes both methods highly
valuable for organic synthesis.
Regarding structure-affinity relationships, the influence of
various lipophilic and hydrophilic functional groups as well as of
different heterocycles on the binding affinity of the bisquaternary
salts was investigated. Notably, molecular structures allowing for
additional lipophilic interactions were found to play a vital role in
the binding event. In line with this observation, lipophilic 3-phenyl-
compounds was
characterization data of the described compounds.
> 95%. See the Supporting Information for the
The following compounds were prepared according to literature:
Propane-1,3-diyl
(21),[43,44]
bis(trifluoromethanesulfonate)
2-tert-
[
27]
2-(pyridin-3-yl)propan-2-ol (9),[33] rac-nicotine (rac-
butylpyridine (17),
26),[ 1-(tert-butyl)-1H-imidazole (41)[29] and the 4-tert-butyl substituted
pyridine derivatives 4-(tert-butyl)-3-methylpyridine (33), 4-(tert-butyl)-3-
methoxypyridine (31), 4-(tert-butyl)-3-chloropyridine (28), 3-bromo-4-(tert-
butyl)pyridine (29), 4-(tert-butyl)pyridine-3-carbonitrile (30), ethyl 4-(tert-
28]
butyl)pyridine-3-carboxylate
chloride (27), 4-(tert-butyl)-3-phenylpyridine (34), 4-(tert-butyl)-N,N-
dimethylpyridine-3-carboxamide (32), and 4-(tert-butyl)-3-ethynylpyridine
(35).[30]
(36),
4-(tert-butyl)-3-fluoropyridin-1-ium
General Procedures
4-tert-butyl substituted bispyridinium compound 3h (PTM0022)
was found to possess a significantly improved binding affinity as
compared to MB327.
Synthesis of symmetric bispyridinium compounds by N-alkylation
with 21 (GP1): 21 (1.0ꢀeq.) was carefully added to a solution of the
corresponding pyridine derivative (2.5ꢀeq.) in CH
the neat pyridine derivative (2.5 eq.) at room temperature. The resulting
mixture was stirred at 50ꢀ°C for the indicated period and residual CH Cl
2 2
Cl (0.8 mL/mmol) or to
Further, differences in binding affinities were found between
2
polar (OMe, NMe , COOEt, etc.) and apolar (C≡CH, Me, i-Pr, etc.)
2
2
substituted compounds, generally revealing higher binding
9
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ChemMedChem
10.1002/cmdc.201800325
FULL PAPER
was removed in vacuo if necessary. The residue was finally recrystallized
methoxypyridin-1-ium) bis(trifluoromethanesulfonate) (2c, colorless solid,
from the given solvent.
84%), 1,1'-(propane-1,3-diyl)bis[3-(dimethylamino)pyridin-1-ium] bis-
(
1
trifluoromethanesulfonate) (2d, yellowish solid, 47%), rac-{1,1'-(propane-
,3-diyl)bis[3-(1-methylpyrrolidin-2-yl)pyridin-1-ium]} bis(trifluoromethane-
Synthesis of symmetric bispyridinium compounds by N-alkylation
with 16 (GP2): 16 (1.0ꢀeq.) was added to a solution of the corresponding
aromatic nitrogen heterocycle (2.5ꢀeq.) in MeCN (0.8ꢀmL/mmol) and the
resulting mixture was heated to 90ꢀ°C (150ꢀW) in a sealed microwave
reactor for the indicated period. The solvent was removed in vacuo and
the residue was recrystallized from the given solvent.
sulfonate) (2e, yellowish solid, 72%).
Compounds of type III (3a–l): 1,1'-(Propane-1,3-diyl)bis[4-(tert-butyl)-3-
fluoropyridin-1-ium] bis(trifluoromethanesulfonate) (3a, colorless solid,
8
5%), 1,1'-(propane-1,3-diyl)bis[4-(tert-butyl)-3-chloropyridin-1-ium] bis-
trifluoromethanesulfonate) (3b, colorless solid, 81%), 1,1'-(propane-1,3-
diyl)bis[3-bromo-4-(tert-butyl)pyridin-1-ium] bis(trifluoromethanesulfonate)
3c, colorless solid, 99%), 1,1'-(propane-1,3-diyl)bis[4-(tert-butyl)-3-
cyanopyridin-1-ium] bis(trifluoromethanesulfonate) (3d, slightly beige solid,
5%), 1,1'-(propane-1,3-diyl)bis[4-(tert-butyl)-3-methoxypyridin-1-ium]
bis(trifluoromethanesulfonate) (3e, colorless solid, 83%), 1,1'-(propane-
,3-diyl)bis[4-(tert-butyl)-3-(dimethylcarbamoyl)-pyridin-1-ium] bis-
trifluoromethanesulfonate) (3f, colorless solid, 77%), 1,1'-(propane-1,3-
diyl)bis[4-(tert-butyl)-3-methylpyridin-1-ium] bis(trifluoromethanesulfonate)
(3g, colorless solid, 89%), 1,1'-(propane-1,3-diyl)bis[4-(tert-butyl)-3-
phenylpyridin-1-ium] bis(trifluoromethanesulfonate) (3h, colorless solid,
9%), 1,1'-(propane-1,3-diyl)bis[4-(tert-butyl)-3-ethynylpyridin-1-ium] bis-
trifluoromethanesulfonate) (3i, colorless solid, 91%), 1,1'-(propane-1,3-
diyl)bis[4-(tert-butyl)-3-(ethoxycarbonyl)pyridin-1-ium] bis(trifluoro-
methanesulfonate) (3j, colorless solid, 98%), 1,1'-(propane-1,3-diyl)bis[4-
tert-butyl)-2-methylpyridin-1-ium] bis(trifluoromethanesulfonate) (3k,
(
Synthesis of 2- and 3-alkylpyridines by cuprate mediated substitution
reactions of 2- and 3-bromopyridines (GP3): The corresponding
bromopyridine derivative (1.0 eq.) was added dropwise to a suspension of
the respective dialkylcuprate (4.0 eq., prepared according to GP4) in THF
or Et O at –78 °C. The reaction mixture was stirred for 2ꢀh at –78 °C before
2
being allowed to stir for additional 15ꢀh at room temperature. The mixture
was cooled to 0ꢀ°C and the reaction was quenched by addition of saturated
(
8
1
(
aqueous NH
4 3
Cl/conc. NH (1:1, 8 mL/mmol). The mixture was filtrated
over celite® and the residue was alternately washed with Et
2
O
(
3ꢀxꢀ5 mL/mmol) and saturated aqueous NaCl (2ꢀxꢀ5ꢀmL/mmol). The layers
9
(
were separated, and the organic phase was washed with saturated
aqueous NaCl solution (10ꢀmL/mmol). The combined aqueous layers were
extracted with Et
2
O (3ꢀxꢀ10ꢀmL/mmol) and the combined organic layers
4
were dried over MgSO . The solvent was carefully removed under reduced
(
pressure (40ꢀ°C bath temperature, 450ꢀmbar) and the resulting crude
colorless solid, 82%), 4-(tert-butyl)-1-{3-[4-(tert-butyl)-3-carboxypyridin-1-
ium-1-yl]propyl}-pyridin-1-ium-3-carboxylate trifluoromethanesulfonate (3l,
colorless solid, 66%).
product was purified in a first step by FC and subsequently by a vacuum
–2
distillation (5∙10 mbar at 200ꢀ°C). The pure alkylpyridine was obtained as
a colorless liquid.
Compounds of type IV (4a–e): 2,2'-(Propane-1,3-diyl)bis(isoquinolin-2-
ium) diiodide (4a, yellow solid, 86%), 1,1'-(propane-1,3-diyl)bis[3-(tert-
butyl)pyrazin-1-ium] diiodide (4b, yellow powder, 78%), 3,3'-(propane-1,3-
diyl)bis(thiazol-3-ium) diiodide (4c, beige solid, 87%), 3,3'-(propane-1,3-
diyl)bis(1-methyl-1H-imidazol-3-ium) diiodide (4d, colorless solid, 92%),
Preparation of (i-Pr)
Bu) CuCN(MgCl) (GP4): The corresponding Grignard reagent (solution
in Et O or THF, 2.0ꢀeq.) was added to a suspension of CuCN (1.0ꢀeq.) in
Et O or THF (2.0–4.5 mL/mmol) at –78ꢀ°C and the resulting mixture was
stirred for 45ꢀmin at –78ꢀ°C, for 5 min at room temperature and again for
0ꢀmin at –78ꢀ°C.
2 2 2 2
9
CuCN(MgCl) , (t-Bu) CuCN(MgCl) and (d -t-
2
2
2
2
3,3'-(propane-1,3-diyl)bis[1-(tert-butyl)-1H-imidazol-3-ium] diiodide (4e,
1
colorless solid, 82%).
Preparation of t-BuCu(CN)ZnBr∙2LiCl: t-BuZnBr (0.5ꢀ
1
1
M
solution in THF,
1.0ꢀmmol, 22.0ꢀmL, 1.0ꢀeq.) was added to a solution of CuCN (995ꢀmg,
1.0ꢀmmol, 1.0ꢀeq.) and LiCl (933ꢀmg, 22.0ꢀmmol, 2.0ꢀeq.) in THF (50 mL)
cuprate suspension was stirred for
2
2
Deuterated compounds: 4-(2-[ H
3 6
]Methyl[1,1,1,3,3,3- H ]propan-2-
yl)pyridine (48, colorless liquid, 31%), 1,1'-(propane-1,3-diyl)bis[4-(2-
]propan-2-yl)pyridin-1-ium]
methanesulfonate) (49, colorless solid, 81%), diethyl [ H
2
2
[
H
3
]methyl[1,1,1,3,3,3- H
6
bis(trifluoro-
at –10ꢀ°C and the resulting 0.15
0ꢀmin at 0 and –78ꢀ°C each.
M
2
2
]malonate (43,
1
2
2
6 6
colorless liquid, 40%), [ H ]propane-1,3-diol (44) and [ H ]propane-1,3-diyl
bis(trifluoromethanesulfonate) (45, grayish and turbid oil, 58%), 1,1'-
2
(
[ H
6
]propane-1,3-diyl)bis[4-(tert-butyl)pyridin-1-ium] bis(trifluoromethane-
Synthesized compounds
sulfonate) (46, colorless solid, 77%).
Pyridine derivatives: 3-tert-Butylpyridine (8, colorless liquid, 43%), 3-
isopropylpyridine (11, colorless and highly volatile liquid, 51%), 2-
isopropylpyridine (6, colorless liquid, 20%), 4-isopropylpyridine (13, clear
and highly volatile liquid, 33%), 4-(tert-butyl)-2-methylpyridine (15,
colorless liquid, 47%).
Biological Evaluation
2
General Procedure for [ H
6
]MB327 MS Binding Assays: Membranes
from frozen Torpedo californica electroplaque tissue were prepared as
–1
previously reported and stored in storage buffer (120 mmol L NaCl, 5
Compounds of type
butyl)pyridin-1-ium] bis(trifluoromethanesulfonate) (1a, colorless solid,
3%), 1,1'-(propane-1,3-diyl)bis[3-(tert-butyl)pyridin-1-ium] diiodide (1b,
I
(1a–h): 1,1'-(Propane-1,3-diyl)bis[2-(tert-
–1
–1
–1
2 4 2 4
mmol L KCl, 8.05 mmol L Na HPO , 1.95 mmol L NaH PO , pH
.4).[24] Total protein concentration was determined by the bicinchoninic
7
7
[45]
acid method using bovine serum albumin as standard.
All binding
yellow crystals, 79%), 1,1'-(propane-1,3-diyl)bis(2-isopropylpyridin-1-ium)
bis(trifluoromethanesulfonate) (1c, colorless solid, 77%), 1,1'-(propane-
[24]
experiments were performed as described previously, but in absence of
CaCl during incubation. For saturation and competition experiments,
2
1,3-diyl)bis(3-isopropylpyridin-1-ium) bis(trifluoromethanesulfonate) (1d,
aliquots of the membrane preparation (60–120 µg protein per sample)
colorless solid, 88%), 1,1'-(propane-1,3-diyl)bis(4-isopropylpyridin-1-ium)
bis(trifluoromethanesulfonate) (1e, colorless solid, 81%), 1,1'-(propane-
2
were incubated with [ H
6
]MB327 (46) as marker (saturation: 2–160 µmol
L–1, competition: 10 µmol L–1) in triplicates in storage buffer in a total
volume of 125 µL for 2 h at 25 °C. In competition experiments the
1,3-diyl)bis(3,5-dimethylpyridin-1-ium) bis(trifluoromethanesulfonate) (1f,
colorless solid, 69%), 1,1'-(propane-1,3-diyl)bis(2,6-dimethylpyridin-1-ium)
bis(trifluoromethanesulfonate) (1g, colorless solid, 75%), 1,1'-(propane-
concentrations of the competitors ranged from 100 nmol L to 1 mmol L–
–1
1.
After incubation, binding samples were processed exactly as previously
1,3-diyl)bis(2,4,6-trimethylpyridin-1-ium)
bis(trifluoromethanesulfonate)
[22]
described. In saturation experiments, the maximum density of binding
(
1h, colorless solid, 81%).
sites (Bmax) and the equilibrium dissociation constant (K
from saturation isotherms calculated for specific binding employing the
one-site – specific binding” regression tool (Prism v. 5.0, GraphPad
Software, La Jolla, CA, USA). Competition curves were analyzed with the
one site–fit Ki” regression tool fixing top and bottom level of the sigmoidal
competition curves to total binding (in absence of competitor, n = 3) and
d
) were determined
Compounds of type II (2a–e): 1,1'-(Propane-1,3-diyl)bis(2-
methoxypyridin-1-ium) bis(trifluoromethanesulfonate) (2a, colorless solid,
“
67%), 1,1'-(propane-1,3-diyl)bis(3-methoxypyridin-1-ium) bis(trifluoro-
“
methanesulfonate) (2b, colorless solid, 83%), 1,1'-(propane-1,3-diyl)bis(4-
1
0
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ChemMedChem
10.1002/cmdc.201800325
FULL PAPER
nonspecific binding (determined by heat denaturation, n = 3). For statistical
comparisons, data were examined by means of a t-test (two sided, α =
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Synthetic procedures and analytical characterization of all compounds as
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1
1
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10.1002/cmdc.201800325
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Entry for the Table of Contents
Toward potential antidotes against organophosphate
poisoning: A series of new bisquaternary compounds was
designed, synthesized and characterized for their binding affinity
toward the nicotinic acetylcholine receptor (nAChR) using our
recently developed MS Binding Assays. New binders and
important structural motives were identified, thus laying the
groundwork for structure affinity relationship guided drug design
of potential nAChR resensitizers.
1
2
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