molecules
Article
One–Pot Phosphate-Mediated Synthesis of Novel
1,3,5-Trisubstituted Pyridinium Salts: A New Family
of S. aureus Inhibitors
Thomas Pesnot 1 , Markus C. Gershater 2 , Martin Edwards 2 , John M. Ward 2 and Helen C. Hailes 1, *
1
2
*
Department of Chemistry, University College London, Christopher Ingold Laboratories,
20 Gordon Street, London WC1H 0AJ, UK; [email protected]
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering,
University College London, Bernard Katz Building, Gower Street, London WC1E 6BT, UK;
[email protected] (M.C.G.); [email protected] (M.E.); [email protected] (J.M.W.)
Correspondence: [email protected]; Tel.: +44-207-679-4654
Academic Editor: Margaret A. Brimble
Received: 17 March 2017; Accepted: 9 April 2017; Published: 12 April 2017
Abstract: Polysubstituted pyridinium salts are valuable pharmacophores found in many biologically
active molecules. Their synthesis typically involves the use of multistep procedures or harsh reaction
conditions. Here, we report water-based phosphate mediated reaction conditions that promote the
condensation of arylacetaldehydes with amines to give 1,3,5-pyridinium salts. The reaction, carried
out at pH 6, provides conditions suitable for the use of less stable aldehydes and amines in this
Chichibabin pyridine condensation. The evaluation of selected 1,3,5-trisubstituted pyridinium salts
highlighted that they can inhibit the growth of S. aureus in the low µg/mL range. The synthetic
accessibility of these compounds and preliminary growth inhibition data may pave the way towards
the discovery of new anti-bacterials based on the 1,3,5-trisubstituted pyridinium scaffold.
Keywords: heteroaromatic synthesis; Chichibabin reaction; synthesis in water; pyridinium salts;
antibacterial activity
1. Introduction
Polysubstituted pyridine and pyridinium salts such as the coenzymes NAD+ and NADP+ [1]
are involved in many essential biochemical processes. They are also part of other biologically
active natural products such as juliprosine [2], the antibacterial alkaloid ficuseptine [3–5], and the
neurotoxic metabolite 1-methyl-4-phenylpyridinium (MPP+ ) [6,7] (Figure 1). In addition, synthetic
pyridinium species have been developed to facilitate gene delivery [8], or act as platelet activation
antagonists [9], and benzylidenehydrazinyl pyridiniums have been investigated as antimicrobial
agents [10]. The synthesis of polysubstituted pyridiniums however typically involves multistep
procedures, or harsh reaction conditions, and the development of rapid and mild methodologies
for the facile preparation of new polysubstituted pyridinium analogues is sought after.
The Chichibabin reaction for the synthesis of polysubstituted pyridines was first reported nearly
100 years ago [11]. The reaction involves the condensation of aldehydes with an amine to yield
pyridiniums in a single synthetic step. Initially, the reaction required elevated temperatures and
pressure, or acid catalysis [12–17], but the intrinsic instability of aldehydes under these forcing
conditions often resulted in product yields that were at best mediocre.
Molecules 2017, 22, 626; doi:10.3390/molecules22040626
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Figure
Figure 1.
1. Examples
Examples of
of biologically
biologically active
active pyridinium
pyridinium salts.
salts.
Figure
1.
Examples
of
biologically
active
pyridinium
salts.
Typically, three products can be isolated from the acid-mediated condensation of amines with
Typically,
three products
products can
can be
be isolated
isolated from
from the
the acid-mediated
acid-mediated condensation of amines with
Typically, three
acetaldehydes in the Chichibabin reaction (Scheme 1); a 1,2,3,5-tetrasubstituted pyridinium 1 (often the
acetaldehydes in the Chichibabin reaction (Scheme 1); a 1,2,3,5-tetrasubstituted pyridinium 1 (often the
major product), which is formed via the auto-oxidation of the product 1,2,3,5-dihydropyridinium 2,
major product), which is formed via the auto-oxidation of the product 1,2,3,5-dihydropyridinium
1,2,3,5-dihydropyridinium 2,
2,
and 1,3,5-trisubstituted pyridinium salts 3 (a minor product often referred to as the ‘abnormal’
and 1,3,5-trisubstituted
1,3,5-trisubstituted pyridinium
pyridinium salts
salts 33 (a
(a minor
minor product
product often
often referred
referred to
to as
as the
the ‘abnormal’
‘abnormal’
Chichibabin product).
Chichibabin product).
Scheme 1. Three products 1–3 isolated from the acid-mediated Chichibabin reaction.
Scheme
Chichibabin reaction.
reaction.
Scheme 1.
1. Three
Three products
products 1–3
1–3 isolated
isolated from
from the
the acid-mediated
acid-mediated Chichibabin
Triflate salts of lanthanides have been used in room temperature reactions to promote the
Triflate salts of lanthanides have been used in room temperature reactions to promote the
Triflate condensation
salts of lanthanides
been amines
used inand
room
temperaturegiving
reactions
the
Chichibabin
reactionhave
between
acetaldehydes,
1, 2, to
or promote
3 in varying
Chichibabin condensation reaction between amines and acetaldehydes, giving 1, 2, or 3 in varying
Chichibabin
reaction
between amines
and acetaldehydes,
giving 1, 2, orin3 ain50varying
amounts
[18].condensation
More recently,
the condensation
of benzylamine
and arylacetaldehydes
mol %
amounts [18]. More recently, the condensation of benzylamine and arylacetaldehydes in a 50 mol %
amountsof
[18].
More recently,
thewater
condensation
benzylamine
and
arylacetaldehydes
inpyridinium
a 50 mol %
solution
ytterbium
triflate in
providedof
the
first report of
a selective
synthesis of
solution of ytterbium triflate in water provided the first report of a selective synthesis of pyridinium
solution
of ytterbium
triflate
in water
provided
the Lewis
first report
of a selective
of pyridinium
salts
3 [19–21].
However,
the use
of such
rare-earth
acid catalysts
raisessynthesis
sustainability
and cost
salts 3 [19–21]. However, the use of such rare-earth Lewis acid catalysts raises sustainability and cost
salts 3 [19–21].
However,
the use
such
rare-earth
Lewis acid
raisesofsustainability
and cost
issues.
Glacial acetic
acid [21]
wasofalso
reported
to promote
thecatalysts
condensation
phenethylamine
and
issues. Glacial acetic acid [21] was also reported to promote the condensation of phenethylamine and
issues.
Glacial
acetic
acid
[21]
was
also
reported
to
promote
the
condensation
of
phenethylamine
phenylacetaldehyde to the pyridinium 3, but such harsh reaction conditions are unsuitable for
phenylacetaldehyde to the pyridinium 3, but such harsh reaction conditions are unsuitable for
and phenylacetaldehyde
to the pyridinium
3, but such harsh reaction conditions are unsuitable for
intrinsically
unstable substrates
such as arylacetaldehydes.
intrinsically unstable substrates such as arylacetaldehydes.
intrinsically
unstable
substrates
such
as
arylacetaldehydes.
In recent studies we have reported that phosphate can catalyse the Pictet-Spengler condensation
In recent studies we have reported that phosphate can catalyse the Pictet-Spengler condensation
In recent
studies wewith
have amines
reportedto
that
phosphate
can catalyse the Pictet-Spengler
condensation
reaction
of aldehydes
give
tetrahydroisoquinoline
alkaloids (THIAs)
[22–24].
reaction of aldehydes with amines to give tetrahydroisoquinoline alkaloids (THIAs) [22–24].
reaction ofwas
aldehydes
aminesthe
toreaction
give tetrahydroisoquinoline
(THIAs)
[22–24].
Phosphate
proposedwith
to catalyse
by favouring formationalkaloids
of the imine
intermediate,
Phosphate was proposed to catalyse the reaction by favouring formation of the imine intermediate,
Phosphate
was
proposed
to
catalyse
the
reaction
by
favouring
formation
of
the
imine
intermediate,
and participating in the phenolic deprotonation. Imine formation and activation are also essential to
and participating in the phenolic deprotonation. Imine formation and activation are also essential to
andChichibabin
participatingpyridine
in the phenolic
deprotonation.
Imine formation
andthat
activation
are also
essential
to
the
synthesis,
and it was therefore
anticipated
phosphate
might
promote
the Chichibabin pyridine synthesis, and it was therefore anticipated that phosphate might promote
synthesis,
and it was in
therefore
that phosphate
the Chichibabin
synthesis of pyridine
polysubstituted
pyridiniums
water. anticipated
Here we report
the use ofmight
mild promote
aqueous
the synthesis of polysubstituted pyridiniums in water. Here we report the use of mild aqueous
the synthesis of polysubstituted
pyridiniums
water.toHere
we report thesalts
use 3ofand
mild
aqueous
phosphate-based
reaction conditions
to provideinaccess
1,3,5-pyridinium
outline
the
phosphate-based reaction conditions to provide access to 1,3,5-pyridinium salts 3 and outline the
phosphate-based
reactionThe
conditions
provide access
to 1,3,5-pyridinium
salts 3growth
and outline
the
versatility
of the reaction.
resultingto
compounds
were evaluated
as new bacterial
inhibitors
versatility of the reaction. The resulting compounds were evaluated as new bacterial growth inhibitors
versatility
of
the
reaction.
The
resulting
compounds
were
evaluated
as
new
bacterial
growth
inhibitors
against S. aureus.
against S. aureus.
against S. aureus.
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2. Results and Discussion
We have
have recently
recently reported
reported aa phosphate
phosphate mediated
mediated biomimetic
biomimetic Pictet-Spengler
Pictet-Spengler synthesis of
THIAs [22]. The mild reaction conditions allowed even the least
least stable
stable arylacetaldehydes
arylacetaldehydes to generate
THIAs in
in good
good yields
yields [22–24].
[22–24]. This
This biomimetic
biomimetic Pictet-Spengler
Pictet-Spengler condensation
condensation reaction is selective
selective
towards the amine component,
component, requiring
requiringaa phenethylamine
phenethylaminesubstrate
substratethat
thatisismeta-substituted
meta-substitutedwith
witha
astrong
strong
electron-donating
group
(e.g.,
a
hydroxyl
group)
such
as
in
3-hydroxyphenethylamine.
electron-donating group (e.g., a hydroxyl group) such as in 3-hydroxyphenethylamine. The
The
condensation
of this
phenethylamine
for example
an aldehyde
such
as phenylacetaldehyde
condensation
of this
phenethylamine
for example
withwith
an aldehyde
such
as phenylacetaldehyde
4a
4a
in potassium
phosphate
(KPi)
buffergave
gavethe
theTHIA
THIA55(Scheme
(Scheme2).
2).Chichibabin
Chichibabin and
and Pictet-Spengler
in potassium
phosphate
(KPi)
buffer
condensations both involve amine and aldehyde components, imine formation and activation, and
therefore by analogy with the Pictet-Spengler reaction it was anticipated that the Chichibabin reaction
could be promoted
promoted by
by phosphates.
phosphates. This hypothesis was investigated
investigated by reacting
reacting phenylacetaldehyde
phenylacetaldehyde 4a
with tyramine
tyramine (4-hydroxyphenethylamine)
(4-hydroxyphenethylamine) 6a (unreactive under biomimetic Pictet-Spengler conditions)
in a 1.2:1 ratio (as reported for the synthesis of THIAs) in KPi
C for 12
KPi buffer
buffer (pH
(pH 6),
6), at
at 60
60 ◦°C
12 h.
h. As expected
no THIA
was
formed,
but
several
compounds
were
detected
in
trace
quantities,
with
the
mainthe
product
THIA was formed, but several compounds were detected in trace quantities, with
main
of
the reaction
yield)
being
thebeing
‘abnormal’
Chichibabin
pyridinium
salt 3a (Scheme
product
of the (5%
reaction
(5%
yield)
the ‘abnormal’
Chichibabin
pyridinium
salt 3a2).
(Scheme 2).
Scheme 2.
2. Products
of of
4a 4a
with
either
tyramine
6a 6a
or
Scheme
Products3a3aand
and5 5resulting
resultingfrom
fromthe
thecondensation
condensation
with
either
tyramine
3-hydroxyphenethylamine,
respectively.
Reagents
and conditions:
(i) 3-hydroxyphenethylamine,
0.1 M
or
3-hydroxyphenethylamine,
respectively.
Reagents
and conditions:
(i) 3-hydroxyphenethylamine,
KPi,MpH
6, pH
60 °C,
12 ◦h;
0.16a,
M 0.1
KPi,MpH
6, pH
60 °C,
12 ◦h.
0.1
KPi,
6, 60
C,(ii)
12 6a,
h; (ii)
KPi,
6, 60
C, 12 h.
In order to optimise reaction conditions to favour formation of the pyridinium product 3a,
In order to optimise reaction conditions to favour formation of the pyridinium product 3a, the ratio
the ratio of 4a to 6a was increased from 1.2:1 to 5:1 to account for the stoichiometry of the reaction,
of 4a to 6a was increased from 1.2:1 to 5:1 to account for the stoichiometry of the reaction, and a range
and a range of buffers were screened at 0.1 M (pH 6) as media for the reaction (Table 1).
of buffers were screened at 0.1 M (pH 6) as media for the reaction (Table 1).
Table 1.
1. Influence
Influence of
of buffer
buffer conditions
conditions on
on the
the formation
formation of
of 3a
3a (Scheme
(Scheme 2ii)
2ii)11..
Table
1
Entry
Buffer
Yield of 3a
Entry
Buffer
Yield of 3a
1
KH2PO4 (KPi)
48%
KH2 POGlc-1-P
48% 40%
4 (KPi)
2 1
2
Glc-1-P
40%
3
PPi
23%
3
PPi
23%
4 4
UMP
UMP
53% 53%
5 5
B(OH)
3
<1%
B(OH)
<1%
3
6
HEPES
3%
6
HEPES
3%
Tris
<1%
7 7
Tris
<1%
8
water only
<1%
8
water only
<1%
◦
Reaction conditions: 4a (5 equiv.), 6a (1 equiv.), 0.1 M buffer at pH 6, 60 C, 12 h. Yields of 3a were determined by
1 Reaction conditions: 4a (5 equiv.), 6a (1 equiv.), 0.1 M buffer at pH 6, 60 °C, 12 h. Yields of 3a were
HPLC
analysis.
determined by HPLC analysis.
The four phosphate-based buffers tested (Table 1, entries 1–4) catalysed the production of
The four phosphate-based buffers tested (Table 1, entries 1–4) catalysed the production of
pyridinium salt 3a, whereas other buffers or water alone (Table 1, entries 5–8) did not significantly
pyridinium salt 3a, whereas other buffers or water alone (Table 1, entries 5–8) did not significantly
promote the reaction. These results suggested that phosphates promote not only the synthesis of THIAs,
promote the reaction. These results suggested that phosphates promote not only the synthesis of THIAs,
but also that of 1,3,5-trisubstituted pyridinium salts 3. All four phosphates, glucose-1-phosphate
but also that of 1,3,5-trisubstituted pyridinium salts 3. All four phosphates, glucose-1-phosphate
(Glc-1-P), inorganic pyrophosphate (PPi), uridine 50 -phosphate (UMP) and inorganic phosphate (Pi)
(Glc-1-P), inorganic pyrophosphate (PPi), uridine 5′-phosphate (UMP) and inorganic phosphate (Pi)
are naturally abundant and essential to cell survival. They are involved in buffering cellular pH, for
Molecules 2017, 22, 626
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Molecules 2017, 22, 426
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are naturally abundant and essential to cell survival. They are involved in buffering cellular pH,
storing
genetic
information
(UMP(UMP
and other
nucleotide
building
blocksblocks
for DNA
and RNA)
and
for storing
genetic
information
and other
nucleotide
building
for DNA
and RNA)
transferring
biochemical
information
(PPi
is
a
by-product
of
the
hydrolysis
of
ATP
mediated
by
kinases).
and transferring biochemical information (PPi is a by-product of the hydrolysis of ATP mediated
Hence,
the
synthesis
of4261,3,5-trisubstituted
pyridiniums, as
well as THIAs,
is likely
to4 of
proceed
by
kinases).
Hence,
synthesis of 1,3,5-trisubstituted
pyridiniums,
as well
as THIAs,
to
Molecules
2017, 22,the
ofat
13 low
Molecules
2017,
22,
426
13 is4 likely
Molecules 2017, 22, 426
4 of 13
Molecules
2017, 22,
22,mild
426 conditions in vivo. This could explain the occurrence of plant natural
4 of
of 13
13
levels
under
products
Molecules
2017,
426
4
proceed
at
low
levels
under
mild
conditions
in
vivo.
This
could
explain
the
occurrence
of
plant
natural
Molecules 2017,
22, 426
4 of 13
Molecules
2017, 22, 426
4 of 13
Moleculesgenetic
2017,22,
22,426
426
ofRNA)
13 4 of
storing
genetic
information
(UMP
andnucleotide
other nucleotide
blocks
forand
and
and
storing
information
(UMP
and
other
building
blocks for
DNA
RNA)
Molecules
2017, 22, 426
13 or
Molecules
2017,
44and
of
13
such
as the
haouamines
[19].
The
in
vivo
condensation
of building
aldehydes
such
asDNA
allysine
with
itself
products
such
as
the
haouamines
[19].
The
in
vivo
condensation
of
aldehydes
such
as
allysine
storing
genetic
information
(UMP
and
other
nucleotide
building
blocks
for
DNA
and
RNA)
andwith
storing
genetic
information
(UMP
and
other
nucleotide
building
blocks
for
DNA
andmediated
RNA)
and
transferring
biochemical
information
(PPi
is
a
by-product
of
the
hydrolysis
of
ATP
by
kinases).
storing
genetic
information
(UMP
and
other
nucleotide
building
blocks
for
DNA
and
RNA)
and
transferring
biochemical
information
(PPi
is
a
by-product
of
the
hydrolysis
of
ATP
mediated
by
kinases).
storing
genetic
information
(UMP
and
other
nucleotide
building
blocks
for
DNA
and
RNA)
and
storing
genetic
information
(UMP
and
other
nucleotide
building
blocks
for
DNA
and
RNA)
and
with
lysine
has
also
been
reported:
the
resulting
polyfunctional
pyridinium
salt
cross-links
in elastin
transferring
biochemical
information
(PPinucleotide
is a resulting
by-product
of the
hydrolysis
of
ATP
mediated
bycross-links
kinases).
itself
or with
lysine
has
also
been
reported:
the
polyfunctional
pyridinium
salt
storing
genetic
information
(UMP
and
other
building
blocks
for
DNA
and
RNA)
and
transferring
biochemical
information
(PPi
isother
a by-product
by-product
of
the
hydrolysis
of
ATP
mediated
by
kinases).
storing
genetic
information
(UMP
andnucleotide
other of
nucleotide
building
blocks
forand
DNA
and
RNA)
and in
storing
genetic
information
(UMP
and
building
blocks
for
DNA
RNA)
and
Hence,
the
synthesis
of 1,3,5-trisubstituted
asthe
well
asATP
THIAs,
is
likely
to
proceed
at low
transferring
biochemical
information
(PPi
is
the
hydrolysis
of
mediated
by
kinases).
Hence,
the
synthesis
of 1,3,5-trisubstituted
well
as
THIAs,
is
likely
to
proceed
at low
transferring
biochemical
information
(PPi
is aapyridiniums,
by-product
ofas
the
hydrolysis
of
ATP
mediated
by
kinases).
transferring
biochemical
information
(PPi
is apyridiniums,
by-product
of
hydrolysis
of ATP
mediated
by kinases).
Hence,
the
synthesis
of
1,3,5-trisubstituted
pyridiniums,
as
well
as
THIAs,
is
likely
to
proceed
at
low
is
believed
to
be
an
age-related
intermolecular
cross-linking
process
[25,26].
transferring
biochemical
information
(PPi
is
a
by-product
of
the
hydrolysis
of
ATP
mediated
by
kinases).
elastin
is
believed
to
be
an
age-related
intermolecular
cross-linking
process
[25,26].
Hence,
the
synthesis
ofmild
1,3,5-trisubstituted
pyridiniums,
asthe
well
as
THIAs,
is
likely
to
proceed
at low
low
transferring
biochemical
information
(PPi
isexplain
a could
by-product
of THIAs,
the
hydrolysis
ofto
ATP
mediated
byproducts
kinases).
transferring
biochemical
information
(PPi
isvivo.
apyridiniums,
by-product
of
hydrolysis
ofis
ATP
mediated
byproducts
kinases).
levels
under
conditions
in
This
explain
the
of
plant
natural
levels
under
mild
conditions
vivo.
This
could
the
occurrence
of
plant
natural
Hence,
the
synthesis
of
1,3,5-trisubstituted
pyridiniums,
as
as
likely
proceed
at
Hence,
the
synthesis
ofmild
1,3,5-trisubstituted
as well
well
as
likely
to
proceed
at low
Hence,
theconditions
synthesis
ofin
1,3,5-trisubstituted
pyridiniums,
asTHIAs,
welloccurrence
asis
THIAs,
is
likely
to
proceed
attolow
levels
under
conditions
in vivo.
This
could
explain
the
occurrence
of
plant
natural
products
Hence,
the
synthesis
of1,3,5-trisubstituted
1,3,5-trisubstituted
pyridiniums,
asthe
well
as
THIAs,
is
likely
to
proceed
atwith
low
The
reaction
were
further
improved
by
increasing
the
KPi
buffer
concentration
0.25
levels
under
mild
conditions
in
vivo.
This
could
explain
occurrence
of
plant
natural
products
The
reaction
conditions
were
further
improved
by
increasing
the
KPi
buffer
concentration
0.25 M
M
Hence,
the
synthesis
of
1,3,5-trisubstituted
pyridiniums,
as
well
as
THIAs,
is
likely
to
proceed
attolow
Hence,
the
synthesis
of
pyridiniums,
as
well
as
THIAs,
is
likely
to
proceed
at
low
such
as
the
haouamines
[19].
The
in
vivo
condensation
of
aldehydes
such
as
allysine
itself
or
levels
under
mild
conditions
in
vivo.
This
could
explain
the
occurrence
of
plant
natural
products
such
as
the
haouamines
[19].
The
in
vivo
condensation
of
aldehydes
such
as
allysine
with
itself
or
levels under
mild
conditions
in
vivo.
This
could
explain
the
occurrence
of
plant
natural
products
levels
under
mild
conditions
in
vivo.
This
could
explain
the
occurrence
of
plant
natural
products
such
as
the
haouamines
[19].
The
in
vivo
condensation
of
aldehydes
such
as
allysine
with
itself
or
levels
under
mild
conditions
in
vivo.
This
could
explain
the
occurrence
of
plant
natural
products
◦
such
as
the
haouamines
[19].
The
in
vivo
condensation
of
aldehydes
such
as
allysine
with
itself
orin
and
elevating
the
temperature
to
100
°C.
The
reaction
pH
was
however
kept
at
6products
since
more
acidic
levels
under
mild
conditions
in
vivo.
This
could
explain
the
occurrence
of
plant
natural
products
levels
under
mild
conditions
in
vivo.
This
could
explain
the
occurrence
of
plant
natural
with
lysine
has
also
been
reported:
the
resulting
polyfunctional
pyridinium
salt
cross-links
elastin
and
elevating
the
temperature
to
100
C.
The
reaction
pH
was
however
kept
at
6
since
more
acidic
such
as
the
haouamines
[19].
The
in
vivo
condensation
of
aldehydes
such
as
allysine
with
itself
or
with
lysine
has
also
been
reported:
the
resulting
polyfunctional
pyridinium
salt
cross-links
in
elastin
such as the
haouamines
[19].been
The[19].
in vivo
of
aldehydes
such
as allysine
itself
orinitself
such
as the has
haouamines
The condensation
in
condensation
of aldehydes
such
as
allysine
with
or
with
lysine
also
reported:
thevivo
resulting
polyfunctional
pyridinium
saltwith
cross-links
elastin
suchlysine
as is
the
haouamines
[19].
The
in vivo
vivo
condensation
of
aldehydes
such
as
allysine
with
itself
oritself
with
has
also
been
reported:
the
resulting
polyfunctional
pyridinium
salt
cross-links
initself
elastin
such
asalso
theto
haouamines
[19].
The
in
vivo
condensation
ofconditions
aldehydes
such
as allysine
with
or of
such
as
the
haouamines
[19].
The
in
condensation
of
aldehydes
such
as
allysine
with
or
conditions
resulted
in
slower
reaction
rates
and
more
alkaline
favoured
the
polymerisation
believed
be
an
age-related
intermolecular
cross-linking
process
[25,26].
with
lysine
has
been
reported:
the
resulting
polyfunctional
pyridinium
salt
cross-links
in
elastin
is
believed
to
be
an
age-related
intermolecular
cross-linking
process
[25,26].
conditions
resulted
in
slower
reaction
rates
and
more
alkaline
conditions
favoured
the
polymerisation
with
lysine
has
also
been
reported:
the
resulting
polyfunctional
pyridinium
salt
cross-links
in
elastin
with
lysine
has
also
been
reported:
the
resulting
polyfunctional
pyridinium
salt
cross-links
in
elastin
is believed
tobeen
be an
age-related
intermolecular
cross-linking
process salt
[25,26].
with
lysine
has
also
reported:
the
resulting
polyfunctional
pyridinium
cross-links
inelastin
elastin
is
believed
to
be
an
age-related
intermolecular
cross-linking
process
[25,26].
with
lysine
has
also
been
reported:
the
resulting
polyfunctional
pyridinium
salt
cross-links
in
elastin
with
lysine
has
also
been
reported:
the
resulting
polyfunctional
pyridinium
salt
cross-links
in
The
reaction
conditions
were
further
improved
by
increasing
the
KPi
buffer
concentration
to
0.25
M
is
believed
to
be
an
age-related
intermolecular
cross-linking
process
[25,26].
The
reaction
conditions
were
further
improved
by
increasing
the
KPi
buffer
concentration
to
0.25
M
phenylacetaldehyde.
Finally,
thethe
solubility
of
both
amine
and[25,26].
aldehyde
substrates
waswas
enhanced
believed
to The
be an
age-related
intermolecular
cross-linking
process
is believed
be
an
age-related
intermolecular
cross-linking
process
[25,26].
ofisisphenylacetaldehyde.
Finally,
solubility
of both
amine
and
aldehyde
substrates
enhanced
reaction
conditions
were
further
improved
by
increasing
the
KPi
buffer
concentration
M by
believed
toelevating
beconditions
anto
age-related
intermolecular
cross-linking
process
[25,26].
The
reaction
were
further
improved
by
increasing
the
KPi
buffer
concentration
to
0.25
Mto 0.25
is
believed
to
be
age-related
intermolecular
cross-linking
process
[25,26].
is believed
to
be
an
age-related
intermolecular
cross-linking
process
[25,26].
and
thean
temperature
to
100 methanol.
°C.
The
reaction
pH
was
however
at more
6new
since
more
acidic
The
reaction
were
further
improved
by
the
KPi
buffer
concentration
to
0.25
M
and
elevating
theconditions
temperature
to
100were
°C.
The
reaction
pH
was
however
kept
atof
6kept
since
acidic
The
reaction
conditions
were
further
improved
by increasing
increasing
the
KPi
buffer
concentration
to
0.25
Mto 0.25
using
a
1:1
mixture
of
KPi
buffer
with
The
combination
these
conditions
was
The
reaction
conditions
further
improved
by
increasing
the
KPi
buffer
concentration
M
and
elevating
the
temperature
to
100
°C.
The
reaction
pH
was
however
kept
at
6
since
more
acidic
by
using
a
1:1
mixture
of
KPi
buffer
with
methanol.
The
combination
of
these
new
conditions
was
Theconditions
reaction
conditions
were
further
improved
byincreasing
increasing
theKPi
KPibuffer
buffer
concentration
to0.25
0.25M
M
and
elevating
the conditions
temperature
to 100
100
°C. improved
The
reaction
pH was
was
however
kept
at
since
more
acidic
The
reaction
conditions
were
further
improved
by
increasing
the
KPi
buffer
concentration
M
The
reaction
were
further
by
the
concentration
to
resulted
in
slower
reaction
rates
and
more
alkaline
conditions
favoured
the
polymerisation
of
and
elevating
the
temperature
to
°C.
The
reaction
pH
however
kept
at
666kept
since
more
acidic
conditions
resulted
in
slower
reaction
rates
and
more
alkaline
conditions
favoured
the
polymerisation
ofto 0.25
and
elevating
the
temperature
to
100
°C.
The
reaction
pH
was
however
kept
at
since
more
acidic
and
elevating
the
temperature
to
100
°C.
The
reaction
pH
was
however
at
6
since
more
acidic
rewarded
with
the
production
of
the
3a
in
70%
isolated
yield.
Aldehyde
4a
was
then
reacted
with
a
conditions
resulted
in
slower
reaction
rates
and
more
alkaline
conditions
favoured
the
polymerisation
of
rewarded
with
the
production
ofrates
the
3a
in
70%
isolated
yield.
Aldehyde
4aatenhanced
was
then
with
and elevating
elevating
the
temperature
to 100
100
°C.
The
reaction
pH conditions
waspH
however
kept at
at
6polymerisation
since
more
acidic
conditions
resulted
in
slower
reaction
rates
and
more
alkaline
favoured
the
ofreacted
and
elevating
the temperature
toand
100
°C.
The
reaction
was
however
6 was
since
more
acidic
and
the
temperature
to
°C.
The
reaction
pH
was
however
kept
6kept
since
more
acidic
phenylacetaldehyde.
Finally,
the
solubility
of both
amine
and
aldehyde
substrates
enhanced
by
phenylacetaldehyde.
Finally,
the
solubility
ofrates
both
amine
and
aldehyde
substrates
was
by
conditions
resulted
in
slower
reaction
more
alkaline
conditions
favoured
the
polymerisation
of
conditions
resulted
in
slower
reaction
rates
and
more
alkaline
conditions
favoured
the
polymerisation
of
conditions
resulted
in
slower
reaction
and
more
alkaline
conditions
favoured
the
polymerisation
of
phenylacetaldehyde.
Finally,
the
solubility
of
both
amine
and
aldehyde
substrates
was
enhanced
by
range
ofof
(hetero)arylethylamines
6b–6g,
arates
benzylamine
6h,
aliphatic
amines
6i–6l,
and
aromatic
conditions
resulted
inmixture
slower
reaction
rates
and
alkaline
conditions
favoured
the
polymerisation
of
aphenylacetaldehyde.
range
6b–6g,
amore
benzylamine
6h,
aliphatic
6i–6l,
and
aromatic
and
phenylacetaldehyde.
Finally,
the
solubility
ofwith
both
amine
and
aldehyde
substrates
was
enhanced
by
conditions
slower
reaction
and
more
alkaline
conditions
favoured
the polymerisation
of and
conditions
resulted
in
slower
rates
and
more
alkaline
conditions
favoured
the
polymerisation
of
a 1:1resulted
of
KPi
buffer
methanol.
The
combination
of
these
new
conditions
was
Finally,
the
solubility
of
both
amine
and
aldehyde
substrates
was
enhanced
by
using
a using
1:1(hetero)arylethylamines
mixture
of
KPiinreaction
buffer
with
The
combination
theseamines
new
conditions
was
phenylacetaldehyde.
Finally,
the
solubility
ofwith
both
amine
and
aldehyde
substrates
was
enhanced
by
phenylacetaldehyde.
Finally,
the methanol.
solubility
of
both
amine
and of
aldehyde
substrates
was
enhanced
by
using
a amines
1:1 mixture
of
KPi
buffer
methanol.
The
combination
of
these
new
conditions
was
phenylacetaldehyde.
Finally,
the
solubility
of
both
amine
and
aldehyde
substrates
was
enhanced
by
heteroaromatic
6m,
6n
to
form
the
corresponding
1,3,5-trisubstituted
pyridinium
salts
6b–6l
using
a
1:1
mixture
of
KPi
buffer
with
methanol.
The
combination
of
these
new
conditions
was
phenylacetaldehyde.
Finally,
the
solubility
of
both
amine
andof
aldehyde
substrates
was
enhanced
by
phenylacetaldehyde.
Finally,
the
of 3a
both
amine
and
aldehyde
substrates
was
enhanced
by
heteroaromatic
amines
6m,
6nsolubility
towith
form
the
corresponding
1,3,5-trisubstituted
pyridinium
rewarded
the
production
ofin
the
in
70%
isolated
yield.
Aldehyde
4a
was
then
reacted
with
a6b–6l
rewarded
with
production
the
3a
70%
isolated
yield.
Aldehyde
4a
was
then
reacted
with
a salts
using
aa 1:1
mixture
of
KPi
buffer
methanol.
The
combination
these
new
conditions
was
using
1:1
mixture
of
KPi
buffer
with
methanol.
The
combination
of
these
new
conditions
was
using
athe
1:1with
mixture
ofofKPi
buffer
with
methanol.
The
combination
of
these
new
was
rewarded
with
of
the
3aisolated
in
70%
isolated
yield.
Aldehyde
4a
was
thenconditions
reacted
with
a
using
arange
1:1
mixture
of the
KPiproduction
buffer
with
methanol.
Theyield.
combination
of4a
these
new
conditions
was
rewarded
with
the
production
ofKPi
the
3a
in
70%
Aldehyde
was
then
reacted
with
acarboxylates
in
13–72%
isolated
yields
(Table
2).
Functionalities
including
hydroxyls,
halogens
and
using
a
1:1
mixture
of
buffer
with
methanol.
The
combination
of
these
new
conditions
was
using
a
1:1
mixture
of
KPi
buffer
with
methanol.
The
combination
of
these
new
conditions
was
of
(hetero)arylethylamines
6b–6g,
a
benzylamine
6h,
aliphatic
amines
6i–6l,
and
aromatic
and
rewarded
with
the
production
of
the
3a
in
70%
isolated
yield.
Aldehyde
4a
was
then
reacted
with
a
range
of
(hetero)arylethylamines
6b–6g,
a
benzylamine
6h,
aliphatic
amines
6i–6l,
and
aromatic
and
in
13%–72%
isolated
yields
(Table
2).
Functionalities
including
hydroxyls,
halogens
and
carboxylates
rewarded
withof
the
production
of the 3aof
in6b–6g,
70%
isolated
yield.
Aldehyde
4a was
then
reacted
with
a with
rewarded
with
the
production
the
3a
in
70%
isolated
yield.
Aldehyde
4a
was
then
reacted
a
range
(hetero)arylethylamines
a
benzylamine
6h,
aliphatic
amines
6i–6l,
and
aromatic
and
rewarded
with the
thewith
production
of6b–6g,
the
3a
in
70%
isolated
yield.
Aldehyde
4a6i–6l,
was then
then
reacted
with
range
of heteroaromatic
(hetero)arylethylamines
benzylamine
6h,
aliphatic
amines
and
aromatic
and
rewarded
the
production
of
the
3athe
inobserved
70%6h,
isolated
yield.
Aldehyde
4a
was
then
reacted
a or
rewarded
with
production
of
the
3a
70%
isolated
yield.
Aldehyde
4a
was
reacted
with
aa with
amines
6m,
to
form
corresponding
pyridinium
salts
6b–6l
were
well
tolerated.
The
lack
of
reactivity
with
aromatic
amines
such
aniline
6m
range
of
6b–6g,
aainbenzylamine
aliphatic
amines
6i–6l,
and
aromatic
and
heteroaromatic
6m,
6n
to
form
the
corresponding
1,3,5-trisubstituted
pyridinium
salts
6b–6l
were
well
tolerated.
The
lack
of6n
with
aromatic
amines
such
as
aniline
6m
or
range
of (hetero)arylethylamines
(hetero)arylethylamines
6b–6g,
benzylamine
6h,
aliphatic
amines
6i–6l,
and
aromatic
and
range
ofamines
(hetero)arylethylamines
aobserved
benzylamine
6h,1,3,5-trisubstituted
aliphatic
amines
6i–6l,
andas
aromatic
and
heteroaromatic
amines
6m,
6nreactivity
toaa6b–6g,
form
the
corresponding
1,3,5-trisubstituted
pyridinium
salts
6b–6l
range of
ofin
(hetero)arylethylamines
6b–6g,
benzylamine
6h,
aliphatic
amines
6i–6l,
and
aromatic
and
heteroaromatic
amines
6m,
6n
to
form
the
corresponding
1,3,5-trisubstituted
pyridinium
salts
6b–6l
range
of
(hetero)arylethylamines
6b–6g,
a
benzylamine
6h,
aliphatic
amines
6i–6l,
and
aromatic
and
range
(hetero)arylethylamines
6b–6g,
a
benzylamine
6h,
aliphatic
amines
6i–6l,
and
aromatic
and
13–72%
isolated
yields
(Table
2).
Functionalities
including
hydroxyls,
halogens
and
carboxylates
heteroaromatic
amines
6m,
6n
to
form
the
corresponding
1,3,5-trisubstituted
pyridinium
salts
6b–6l
in
13–72%
isolated
yields
(Table
2).
Functionalities
including
hydroxyls,
halogens
and
carboxylates
2-aminobenzimidazole
6n
may
originate
from
their
poor
nucleophilicity
as
well
as
steric
hindrance.
heteroaromatic
amines
6m,
6n
to
form
the
corresponding
1,3,5-trisubstituted
pyridinium
salts
6b–6l
heteroaromatic
amines
6m,(Table
6n to 2).
form
the corresponding
1,3,5-trisubstituted
pyridinium
salts
6b–6l
2-aminobenzimidazole
6nyields
may
originate
from
their poor
nucleophilicity
as well
as
steric
hindrance.
in 13–72% isolated
Functionalities
including
hydroxyls, halogens
and
carboxylates
heteroaromatic
amines
6m,
6n
to
form
theform
corresponding
1,3,5-trisubstituted
pyridinium
salts
6b–6l
in
13–72%
isolated
yields
(Table
2).
Functionalities
including
hydroxyls,
halogens
and
carboxylates
heteroaromatic
amines
6m,
6n the
to
the corresponding
salts6m
6b–6l
heteroaromatic
amines
6m,
6n
to
form
corresponding
pyridinium
salts
6b–6l
were
well
tolerated.
The
lack
of
reactivity
observed
with1,3,5-trisubstituted
aromatic
amines
such
as 6m
aniline
or
were
well
tolerated.
The
lack
of
reactivity
observed
with1,3,5-trisubstituted
aromatic
amines
such
as pyridinium
aniline
or
in
13–72%
isolated
yields
(Table
2).
Functionalities
including
hydroxyls,
halogens
and
carboxylates
in
13–72%
isolated
(Table
2).
Functionalities
including
hydroxyls,
halogens
and
carboxylates
in
13–72%
isolated
yields
(Table
Functionalities
including
hydroxyls,
halogens
and
carboxylates
were
well yields
tolerated.
The
lack
of2).
reactivity
observed
with aromatic
amines
such
as6m
aniline
6m or
in 13–72%
13–72%
isolated
yields
(Table
2).
Functionalities
including
hydroxyls,
halogens
and
carboxylates
were
well
tolerated.
The
lack
of
reactivity
observed
with
aromatic
amines
such
as
aniline
or
in
13–72%
isolated
yields
(Table
2).
Functionalities
including
hydroxyls,
halogens
and
carboxylates
in
isolated
yields
(Table
2).
Functionalities
including
hydroxyls,
halogens
and
carboxylates
2-aminobenzimidazole
6nreactivity
may
originate
from
their
poor
nucleophilicity
asas
well
as
steric
hindrance.
2-aminobenzimidazole
6n
may
originate
from
their
poor
nucleophilicity
as well
as
steric
hindrance.
were
well
tolerated.
The
lack
of
observed
with
aromatic
amines
such
aniline
or
11.6m
were
well
tolerated.
The
lack
of
reactivity
observed
with
aromatic
amines
such
as
aniline
6m
or
were
well
tolerated.
The
lack
of
reactivity
observed
with
aromatic
amines
such
as
aniline
6m
Table
2.
One-step
phosphate
mediated
synthesis
of
1,3,5-pyridiniums
3
2-aminobenzimidazole
6n reactivity
may
originate
from
their
poor
nucleophilicity
well
as3steric
Table
One-step
phosphate
mediated
synthesis
ofamines
1,3,5-pyridiniums
. 6mhindrance.
were well
well
tolerated.
The
lack
of
observed
with
aromatic
such
as such
aniline
or 6m or
2-aminobenzimidazole
6n2.may
may
originate
from
their
poor
nucleophilicity
as well
well
asassteric
steric
hindrance.
were
well tolerated.
lack offrom
reactivity
observed
with aromatic
amines
as 6m
aniline
or
were
tolerated.
The
of
reactivity
observed
with
aromatic
amines
such
as
aniline
or
2-aminobenzimidazole
6n
originate
their
poor
nucleophilicity
as
as
hindrance.
2-aminobenzimidazole
6n lack
mayThe
originate
from
their
poor
nucleophilicity
as well
asassteric
hindrance.
2-aminobenzimidazole
6n
may
originate
from
their
poor
nucleophilicity
well
as
steric
hindrance.
2-aminobenzimidazole
6n
may2.originate
originate
from
their
poor
nucleophilicity
aswell
well3as
as
steric
hindrance.
1.steric hindrance.
1. steric
Table
One-step
phosphate
mediated
synthesis
of 1,3,5-pyridiniums
3hindrance.
2-aminobenzimidazole
6n
may originate
from
their
poor
nucleophilicity
as
well as
Table 2.6n
One-step
phosphate
mediated
synthesis
of 1,3,5-pyridiniums
2-aminobenzimidazole
may
from
their
poor
nucleophilicity
as
1
Table 2. phosphate
One-step phosphate
of 1,3,5-pyridiniums
3 .
1
Table 2.
2. One-step
mediated mediated
synthesis synthesis
of 1,3,5-pyridiniums
1,3,5-pyridiniums
Table
mediated
synthesis
of
333 11...
Table 2. One-step
One-step
phosphatephosphate
mediatedmediated
synthesis synthesis
of 1,3,5-pyridiniums
Table 2. phosphate
One-step
of 1,3,5-pyridiniums
3 1.
1
Table2.
2.One-step
One-step
phosphate
mediatedmediated
synthesissynthesis
of1,3,5-pyridiniums
1,3,5-pyridiniums
Table
2. One-step
phosphate
of 1,3,5-pyridiniums
3 1.
Table
phosphate
mediated
synthesis
of
331..
Amine
Amine
Amine
Amine
Amine
6a
Amine
Amine
6a
6a
Amine
6a
6a
6a
6a
6a
6b
6b
6b
6b
6b
6b
6b
6b
6c6c
6c
6c
6c
6c
6c
6c
6d
6d
6d
6d
6d
6d
6d
6d
6e
6e
6e
6e
6e
6e
6e
6e
6f
6f
6f
6f
6f
6f
6f
6f
R
R
Product Isolated Yield Amine
Product Product
Isolated
Yield
Amine
Isolated
Yield Amine R
Isolated Yield
Amine
Amine R
R Product
Amine
R
R
Product3a
R
Product
Amine R
R Product
R
Product
6a
3a
3a
Amine
R Product
R
6a
3a
3a
3a
6a
3a
6a PhCH2CH2
3a
3b
6b PhCH
2CH2PhCH2CH2 3b
6b
PhCH
2
CH
2
PhCH
CH
3b
22CH22
3b
PhCH
3b
PhCH22CH
CH22PhCH2CH2 3b
6bPhCH
3b
PhCH22CH
CHPhCH
2
6b PhCH
2CH2 3b
2
6c
3c 3c
6c
3c
3c
3c
3c
6c
3c
6c
3c
6d
3d
3d
6d
3d
3d
3d
3d
6d
3d
6d
3d
6e
3e
6e
3e
3e
3e
3e
6e
3e
3e
6e
3e
6f
3f
6f
3f
3f
3f 3f
6f
3f
3f
6f
3f
Product Isolated Yield
Isolated Yield
Yield
Amine
Isolated
70% Amine
Isolated Yield
Amine
Product
Isolated
Yield
Isolated
Yield
Amine
3a
70%
6h
70%
70%
Product
Isolated
Yield
Isolated
Yield
Amine
3a
70%
6h
70%
6h
70%
3a 70%
70%6h
6h
70%
3a 70%
70% 6h
3b 54% 54%54% 6i
3b
54%
54%
6i
54%
6i
54%
3b54%
54%6i
6i
54%
3b 54%
54% 6i
3c 52% 52%52% 6j
3c
52%
6j
52%52%
6j
52%
3c 52%
52%6j
6j
52%
3c 52%
52% 6j
3d 38%
38%6k
38%38%6k
3d
38%
6k
38%38% 38%6k
3d38%
6k
38%
3d 38%
38% 6k
3e 50%
50% 6l
3e
50%
6l
50% 50%
6l
50%50% 50%6l
3e50%
6l
50%
3e 50%
50% 6l
3f 45%
45%6m
3f
45%
6m
45%
6m
45% 45%45%6m
3f 45%
6m
45%
45%
3f 45%
45%6m
Amine
R
6h
R
Amine R
R
6h
6h
Amine
6h R
R
Product Isolated Yield
R
Product Isolated
Isolated
Yield
R Product Product
Yield
Isolated Yield
R
Product
Product
R Product
Product
3h
R Product
3h
3h
3h
6h
3h
3h
6i6h
6i
3i
6i 6i
3i
3i
3i
6i
3i
6i
3i
(CH22))66OH
6j 6j(CH2)6OH(CH
OH3j
6j
(CH2)6OH
3j
(CH22))66OH
OH (CH ) OH
6j (CH
3j
(CH2)6OH (CH22)66OH3j
6j
3j
(CH22))66OH
OH(CH2)6OH3j
6j(CH
6k
H
(CH2)3CO2(CH
H 2)3CO23k
6k(CH
(CH
CO23k
2H
H
6k
(CH22)3CO
2)3CO2H
H
6k(CH
CO23k
2H
3k
(CH
CO2(CH
2H
6k 22))33CO
(CH22))33CO
H
3k
(CH
2)3CO2H
6k
3k
(CH2)3CO2(CH
H 2)3CO2H
6l
3l
6l
3l
6l
3l
3l
6l 6l
3l
6l
3l
6m Ph
Ph 3m
6m
Ph
3m
Ph
6m6m Ph
Ph
3m
Ph
Ph 3m
3m
Ph
6m6m Ph
Ph
Ph 3m
Product Isolated Yield
Isolated
Yield
Isolated
3h Yield
65%
Isolated
Yield
Product
Isolated Yield
Isolated
Yield
3h
65%Yield
65%
3h
65%
Product
Isolated
Isolated
Yield
3h
65%
65%
65%
3h65%
65%
65%
3h3i
65%
65%
3i 72%
72% 72%
3i3i
72%72%
72%
72%
3i 72%
72%
72%
3i 72%
72%
3j 3j
69% 69%
69%
3j
69%
69%
3j
69%
69%
3j 69%
69%
69%
3j 69%
69%
3k 63%
63%
3k3k
63% 63%
63%
63%
63%
3k3k
63%63%
63%
3k 63%
63%
3l 53%
53%
3l
53%
53%
3l
53%
53%
53%
3l3l
53%53%
53%
3l 53%
53%
3m 0%
0%
3m
0%
0%
0%
0%
3m3m
0% 0%
0%
3m
3m 0%
0% 0%
3n 0%
0%
3n
0%
0%
0%
0%
3n3n
0% 0%
0%
3n 0%
0%
6g
3g13% 2
6n
13% 26n
6g
3g
3n
6g
3g
6n
13% 2
6g
3g
6n
3n
13% 22
6g
3g
6n
3n
13%
2
2
6g
3g 3g
6n
3n
6g
3g13% 213%13%
2
6g
6n6n
6g
3g
6n
3n
13%2 2 13%
26n
1 Reaction conditions: 4a
1 Reaction
6g
3g
6n
6g
3g(5amine
6n
3npH
equiv.),
amine
6 (10.25
equiv.),
0.25
M KPi buffer:MeOH
(1:1)
at
13%
conditions: 4a (5 equiv.),
613%
(1
equiv.),
M KPi
buffer:MeOH
(1:1)3n
at pH
6, 100
°C,6, 100 °C,0%
1
Reaction
conditions:
4a (5amine
equiv.),
6 (10.25
equiv.),
0.25
M KPi buffer:MeOH
(1:1)
at pH
Reaction
conditions:
4a (5
(5
equiv.),
(1amine
equiv.),
M KPi
KPi
buffer:MeOH
(1:1)
at
pH
6,h100
100
°C, 6, 100 °C,
2 4a (1.2
equiv.),
amine
6666 (1
equiv.),
0.1
M
buffer
at
60
°C,
12
(Pictet-Spengler
h; equiv.),
(1.2
amine
6 (5
(1
equiv.),
0.1
M 6KPi
buffer
at
pH
6,KPi
60 pH
°C, 6,
12
h at
(Pictet-Spengler
12
h; 2 4a12
Reaction
4a
equiv.),
amine
(1
0.25
M
KPi
buffer:MeOH
(1:1)
pH
6,
°C,
1conditions:
2 4a conditions:
Reaction
conditions:
4a
(5
equiv.),
amine
(1
equiv.),
0.25
M
KPi
buffer:MeOH
(1:1)
at
pH
6,
100
°C,6, 100 °C,
Reaction
4a
equiv.),
amine
(1
equiv.),
0.25
M
buffer:MeOH
(1:1)
at
pH
(1.2
equiv.),
amine
6
(1
equiv.),
0.1
M
KPi
buffer
at
pH
6,
60
°C,
12
h
(Pictet-Spengler
12
h;
1
112
Reaction
conditions:
4a4a
(5(5
equiv.),
amine
(1 equiv.),
equiv.),
0.25
M0.25
KPi
buffer:MeOH
(1:1)
at pH
pH
100
°C,
1(1.2
1Reaction
4a reaction
equiv.),
amine
(1
equiv.),
0.1
M
KPi
buffer
at0.25
pH
6,
60 buffer:MeOH
°C,
12
h (Pictet-Spengler
(Pictet-Spengler
h; 222 4a
conditions:
equiv.),
amine
6(1
(1KPi
M
KPi
(1:1)
at
pH
6, ◦100
°C,h;
Reaction
conditions:
4a
(5amine
equiv.),
amine
6equiv.),
(10.25
equiv.),
M6,
KPi
buffer:MeOH
(1:1)
at
pH
6,100
100
°C,12
Reaction
conditions:
4a
(5
66 (1
M
KPi
buffer:MeOH
(1:1)
at
6,6, 100
°C,
112
conditions).
reaction
(1.2
amine
666 (1
equiv.),
M
buffer
at
60
°C,
h
h;
2 4a
Reaction
conditions:
4a
(5equiv.),
equiv.),
amine
6equiv.),
0.25
MpH
KPi
(1:1)
pH
6,
C,
(1.2
equiv.),
amine
(1
equiv.),
0.1
Mequiv.),
KPi0.1
buffer
at
pH
6,buffer:MeOH
60 pH
°C, 12
12
h (Pictet-Spengler
12
h; 24aconditions).
(1.2
equiv.),
amine
6 (10.1
M
KPi
buffer
at
6,
60
°C,
12 at
h (Pictet-Spengler
12
h; equiv.),
reaction
conditions).
4a12(1.2
(1.2
amine
6 (1
(1 equiv.),
equiv.),
0.1
M KPi
KPi
buffer
at◦buffer
pH 6,
6,at
60pH
°C,6,12
1260hh °C,
(Pictet-Spengler
12 h;
h;22 4a
2equiv.),
reaction
conditions).
2
4a
(1.2
equiv.),
amine
6
(1
equiv.),
0.1
M
KPi
12
h
(Pictet-Spengler
h;
equiv.),
amine
6
0.1
M
buffer
at
pH
60
°C,
(Pictet-Spengler
12
reaction
conditions).
(1.2 amine
equiv.),
(1Mequiv.),
0.1atMpHKPi
pH 6, 60 °C, reaction
12 h (Pictet-Spengler
12
4ah;(1.24a
equiv.),
6 (1amine
equiv.),60.1
KPi buffer
6, 60buffer
C, 12 at
h (Pictet-Spengler
conditions).
reaction
conditions).
reaction
conditions).
reaction
conditions).
In
contrast
to the the
amines,
the reaction
wasselective
highly selective
substrates
reaction
In
contrast
to
theconditions).
amines,
reaction
was highly
towards towards
aldehydealdehyde
substrates
(Table 3).(Table 3).
reaction
conditions).
reaction
conditions).
1
1
1
In to
contrast
to thethe
amines,
the was
reaction
was
highly towards
selective aldehyde
towards aldehyde
substrates
3).
In tyramine
contrast
the
amines,
reaction
highly
selective
substrates
(Table
3).(Tablewas
While
tyramine
6athe
reacted
with
arylacetaldehydes
and
(hetero)arylacetaldehydes,
no reaction
While
6athe
reacted
with
arylacetaldehydes
and
(hetero)arylacetaldehydes,
no reaction
was
In
contrast
to
amines,
the
reaction
was
highly
selective
towards
aldehyde
substrates
(Table
3).
In
contrast
to
the
amines,
the
reaction
was
highly
selective
towards
aldehyde
substrates
(Table
3).
In
contrast
to
amines,
the
reaction
was
highly
selective
towards
aldehyde
substrates
(Table
3).
While
tyramine
6a
reacted
with
arylacetaldehydes
and
(hetero)arylacetaldehydes,
no
reaction
was
In
contrast
towith
theamines,
amines,
the
reaction
was
highly
selective
towards
aldehyde
substrates
(Table
In
contrast
to
the
amines,
the
reaction
was
highly
selective
towards
aldehyde
substrates
(Table
3).
While
tyramine
6a
reacted
with
arylacetaldehydes
and
(hetero)arylacetaldehydes,
no reaction
reaction
was
Inaliphatic
contrast
to
the
amines,
thethe
reaction
was
highly
selective
towards
aldehyde
substrates
(Table
3). 3).
In
contrast
to
the
the
reaction
was
highly
selective
towards
aldehyde
substrates
(Table
3).
observed
aliphatic
aldehydes
with
the
exception
3-methylbutyraldehyde
4r:
ofwas
6a
While
6a
reacted
arylacetaldehydes
and
(hetero)arylacetaldehydes,
no
was
observed
with
aldehydes
with
exception
of
3-methylbutyraldehyde
4r:
of
6a
While
tyramine
6a
reacted
with
arylacetaldehydes
(hetero)arylacetaldehydes,
no reaction
reaction
was
While
tyramine
6awith
reacted
with
arylacetaldehydes
andof
noreaction
reaction
In tyramine
contrast
to
the
amines,
the
reaction
was
highly
selective
towards aldehyde
substrates
(Table
3).
observed
with
aliphatic
aldehydes
with
theand
exception
of(hetero)arylacetaldehydes,
3-methylbutyraldehyde
4r:
reaction
of 6a was
While
tyramine
6a
reacted
with
arylacetaldehydes
and
(hetero)arylacetaldehydes,
no
reaction
was
observed
with
aliphatic
aldehydes
with
the
exception
of
3-methylbutyraldehyde
4r:
reaction
of
6a
While
tyramine
6a
reacted
with
arylacetaldehydes
and
(hetero)arylacetaldehydes,
no
reaction
While
tyramine
6athe
reacted
with
arylacetaldehydes
(hetero)arylacetaldehydes,
noof
reaction
While4rtyramine
6athe
reacted
with
arylacetaldehydes
(hetero)arylacetaldehydes,
noreaction
reaction
was
with
4raliphatic
produced
pyridinium
3rwith
in
a low
yield
ofand
5%ofand
also the pyridinium
1r4r:
(10%
yield).ofwas
The
observed
with
aldehydes
the
exception
of
3-methylbutyraldehyde
6a
with
produced
pyridinium
3rwith
in a low
yield
ofand
5%
and
also
the
pyridinium
1r4r:
(10%
yield).
The
observed
with
aliphatic
aldehydes
with
the
exception
of
3-methylbutyraldehyde
4r:
reaction
of
6a
observed
with
aliphatic
aldehydes
the
exception
3-methylbutyraldehyde
reaction
6a
with
4r
produced
the
pyridinium
3r
in
a
low
yield
of
5%
and
also
the
pyridinium
1r
(10%
yield).
The
While
6a
reacted
with
arylacetaldehydes
(hetero)arylacetaldehydes,
no
reaction
was
observed
with aliphatic
aliphatic
aldehydes
with
thewith
exception
ofand
3-methylbutyraldehyde
4r: reaction
reaction
of
6a
with
4rtyramine
produced
the
pyridinium
3r in
in
low
yield
of
5%
and
also
the
pyridinium
1r to
(10%
yield).
The
observed
with
aldehydes
the
exception
ofthe
3-methylbutyraldehyde
4r:
of 6aof
observed
with
aldehydes
with
the
exception
of
3-methylbutyraldehyde
of
6a
observed
with
aliphatic
aldehydes
the
exception
of
3-methylbutyraldehyde
4r:
reaction
formation
ofpyridinium
1raliphatic
suggested
the
phosphate-mediated
reaction
was
analogous
to(10%
areaction
Chichibabin
with
4r
the
3r
aaawith
low
of
5%
and
pyridinium
1r
(10%
yield).
The
formation
of 4r
1r produced
suggested
that
the
phosphate-mediated
reaction
was
analogous
a Chichibabin
with
4r produced
produced
the
3r that
in
low
yield
of
5%
and
also
the
pyridinium
1r4r:
(10%
yield).
with
the
pyridinium
3r yield
in
a low
yield
ofalso
5%
and
also
the
pyridinium
1r
yield).
The 6a
formation
of pyridinium
1r
suggested
that
the
phosphate-mediated
reaction
was
analogous
to 4r:
a The
Chichibabin
with
4r
produced
the
pyridinium
3r
in
a
low
yield
of
5%
and
also
the
pyridinium
1r
(10%
yield).
The
observed
with
aliphatic
aldehydes
with
the
exception
of
3-methylbutyraldehyde
reaction
of
6a
formation
of
1r
suggested
that
the
phosphate-mediated
reaction
was
analogous
to
a
Chichibabin
with
4r
produced
the
pyridinium
3r
in
a
low
yield
of
5%
and
also
the
pyridinium
1r
(10%
yield).
The
with4r
4r produced
the
pyridinium
3r
inaldehyde
adirected
low
yield
5% the
and
also
pyridinium
1rto
(10%
yield).
The
type
condensation
where
the
directed
reaction
towards
Chichibabin
product
formation
of
that
the
phosphate-mediated
reaction
was
analogous
aa Chichibabin
type
condensation
where
the
aldehyde
theofreaction
towards
either
theeither
Chichibabin
product
with
produced
3r
in phosphate-mediated
a low
yield
of
5%the
and
also
the
pyridinium
1r (10%
yield).
formation
of 1r
1r suggested
suggested
that
the
phosphate-mediated
reaction
was
analogous
tothe
Chichibabin
formation
ofthe
1r pyridinium
suggested
that
the
reaction
was
analogous
to
a Chichibabin
type
condensation
where
the
aldehyde
directed
the
reaction
towards
either
the
Chichibabin
product
formation
of
1r
suggested
that
the
phosphate-mediated
reaction
was
analogous
to
a
Chichibabin
type
condensation
where
the
aldehyde
directed
the
reaction
towards
either
the
Chichibabin
product
with
4r
produced
the
pyridinium
3r
in
a
low
yield
of
5%
and
also
the
pyridinium
1r
(10%
yield).
The
formation
of
1r
suggested
that
the
phosphate-mediated
reaction
was
analogous
to
a
Chichibabin
formation
of
1r
suggested
that
the
phosphate-mediated
reaction
was
analogous
to
a
Chichibabin
1type
via anof
oxidation
reaction
or aldehyde
1,3,5-trisubstituted
pyridiniums
3 viathe
anwas
elimination
step
(Scheme
1).
1 via
an oxidation
reaction
or
1,3,5-trisubstituted
pyridiniums
3 via reaction
an
elimination
step
1).a Chichibabin
type
condensation
where
the
aldehyde
directed
the
reaction
towards
either
Chichibabin
product
The
formation
1r
suggested
that
the
phosphate-mediated
analogous
to
type
condensation
where
the
aldehyde
directed
the
reaction
towards
either
Chichibabin
product
where
the
the
reaction
towards
either
the (Scheme
Chichibabin
product
1 via condensation
an oxidation
reaction
or 1,3,5-trisubstituted
pyridiniums
3 via the
an
elimination
step
(Scheme
1).
type
condensation
wherethe
the
aldehyde
directeddirected
the reaction
reaction
towards
either
the
Chichibabin
product
via an
an
oxidation
reaction
or
1,3,5-trisubstituted
pyridiniums
3 via
via
antowards
elimination
step
(Scheme
1).
type
condensation
where
the
aldehyde
directed
the reaction
either
the(Scheme
Chichibabin
product
condensation
where
aldehyde
directed
the
towards
either
the
Chichibabin
product
formation
of
1r
suggested
that
the
phosphate-mediated
reaction
was
analogous
to
a
Chichibabin
111type
via
oxidation
reaction
or
1,3,5-trisubstituted
pyridiniums
3
an
elimination
step
1).
via an
oxidation
reaction
or
1,3,5-trisubstituted
pyridiniums
3
via
an
elimination
step
(Scheme
1).
1
via
an
oxidation
reaction
or
1,3,5-trisubstituted
pyridiniums
3
via
an
elimination
step
(Scheme
1).
viaan
an1oxidation
oxidation
reactionreaction
or1,3,5-trisubstituted
1,3,5-trisubstituted
pyridiniums
via an
an3elimination
elimination
step (Scheme
(Scheme
1).
via an oxidation
or 1,3,5-trisubstituted
via aneither
elimination
step (Scheme
1).
11 via
reaction
pyridiniums
33 via
step
1).
type
condensation
whereor
the aldehyde
directed
the pyridiniums
reaction
towards
the Chichibabin
product
1 via an oxidation reaction or 1,3,5-trisubstituted pyridiniums 3 via an elimination step (Scheme 1).
Molecules 2017, 22, 626
5 of 13
type condensation where the aldehyde directed the reaction towards either the Chichibabin product 1
5 of 13
or 1,3,5-trisubstituted pyridiniums 3 via an elimination step (Scheme 1).
Molecules
2017, 22, 426reaction
via an oxidation
Molecules 2017, 22, 426
5 of 13
Table
aldehydes 4 11.
Table 3.
3. Versatility
Versatility of the phosphate-mediated pyridinium synthesis towards aldehydes
Table 3. Versatility of the phosphate-mediated pyridinium synthesis towards aldehydes 4 1.
Aldehyde
Aldehyde
Aldehyde
4a
4a4a
4o
4o
4o
R
R
R
PhCH2
PhCH22
PhCH
4-HOC6H4CH2
H44CH
CH22
4-HOC
4-HOC66H
4p
4p
4p
4q
4q
4q
4r
4r4r
4s
4s4s
4-MeOC6H4CH2
4-MeOC66H
H4CH
CH2
4-MeOC
CH(CH43)2 2
3
)
2
CH(CH
CH(CH
C2H35 )2
C
C 2H
H5
Product
Product
Product
3a
3a
3a
3o
3o
3o
Yield
Yield
Yield
70%
70%
70%
66%
66%
66%
3p
3p
3p
42%
42%
42%
3q
3q
3q
3r (1r) 2
22
3r
3r (1r)
(1r)
3s
3s
3s
58%
58%
58%
5(10)%
5(10)%
5(10)%
0%
0%
0%
Reaction conditions: 4 (5 equiv.), amine 6a2(1 5equiv.), 0.25 M KPi buffer:MeOH (1:1) at pH 6, 100 °C,
44 (5
amine 6a (1 equiv.), 0.25 M KPi buffer:MeOH (1:1) at pH 6, 100 °C,
(5 equiv.),
equiv.),
1r. amine 6a (1 equiv.), 0.25 M KPi buffer:MeOH (1:1) at pH 6, 100 ◦ C, 12 h;
12 h; Reaction also yielded
2
2
also yielded
1r.
12 Reaction
h; Reaction
also yielded
1r.
1
1
conditions:
1Reaction
2
Reaction
conditions:
These results are consistent with the recent work published by Baran et al. on the
These
results
with
recent
published
by
etet al.
on
These
results are
are consistent
consistent
with the
the
recent work
worksalts
published
by Baran
Baran
al. that
on the
the
Yb(OTf)
3-mediated
Chichibabin
synthesis
of pyridinium
[19,20]. They
suggested
the
Yb(OTf)
3-mediated Chichibabin synthesis of pyridinium salts [19,20]. They suggested that the
Yb(OTf)
-mediated
Chichibabin
synthesis
of
pyridinium
salts
[19,20].
They
suggested
that
the
lanthanide
3 salt acts as a Lewis acid catalyst and promotes the synthesis of 3, with benzylic group
lanthanide
salt
acts
as
a
Lewis
acid
catalyst
and
promotes
the
synthesis
of
3,
with
benzylic
group
lanthanide
saltvia
acts
as a Lewis
acid catalyst
and promotes
the synthesis
of 3,etwith
benzylic
removal
at C-2
oxidation
and formation
of benzaldehyde.
Alternatively,
Poupon
al. suggested
removal
at C-2 via
and formation
of benzaldehyde.
Alternatively,
Poupon et al.
suggested
group
removal
atoxidation
C-2
oxidation
andpyridinium
formation
of
benzaldehyde.
Poupon
et al.
that
toluene
may
be via
generated
upon
aromatisation
[21].Alternatively,
In this study,
we have
that
toluenethat
may
be generated
upon pyridinium
aromatisation
[21]. [21].
In this
study,
we
have
suggested
toluene
may
be
generated
upon
pyridinium
aromatisation
In
this
study,
we
have
demonstrated that the synthesis of 3 can also be promoted by phosphates in good isolated yields.
demonstrated
that
synthesis
of 3 3can
be
by
iningood
isolated yields.
demonstrated
thatthe
the
synthesis
canalso
also
bepromoted
promoted
byphosphates
phosphates
good
yields.
Phosphate
acid/base
catalysis
canofpromote
keto-enol
equilibria,
proton transfers,
andisolated
eliminations,
Phosphate
acid/base
catalysis
can
promote
keto-enol
equilibria,
proton
transfers,
and
eliminations,
Phosphate
acid/base
catalysis
promote keto-enol
equilibria,Here,
proton
eliminations,
which
typically
occur during
thecan
Chichibabin
pyridine synthesis.
thetransfers,
excellentand
nucleophilic
and
which
typically
occur
during
the
Chichibabin
pyridine
synthesis.
Here,
the
excellent
nucleophilic
and
which
typically
occur
during
the
Chichibabin
pyridine
synthesis.
Here,
the
excellent
nucleophilic
and
leaving group abilities of phosphates may further facilitate the reaction. This concept is reminiscent
leaving
group
abilities
of
phosphates
may
further
facilitate
the
reaction.
This
concept
is
reminiscent
leaving
group abilities
of phosphates
may further
the reaction.
This
is reminiscent
of
investigations
by Sutherland
and co-workers
onfacilitate
the prebiotic
synthesis
of concept
nucleobases
in which
ofofinvestigations
by
Sutherland
and
co-workers
on
the
prebiotic
synthesis
ofofnucleobases
ininwhich
investigations
by
Sutherland
and
co-workers
on
the
prebiotic
synthesis
nucleobases
which
phosphate is described as a general acid/base catalyst as well as a nucleophile activating nitriles
for
phosphate
is
described
as
a
general
acid/base
catalyst
as
well
as
a
nucleophile
activating
nitriles
for
phosphate is
described asreactions
a general[27].
acid/base
aselimination
well as a nucleophile
activating
for
subsequent
condensation
Here, incatalyst
the final
step producing
3, lossnitriles
of proton
subsequent
condensation
reactions
[27].
Here,
ininthe
elimination
step
3,3,loss
ofofproton
subsequent
condensation
reactionsand
[27].the
Here,
thefinal
final
elimination
stepproducing
lossat
proton
3-H
can be assisted
by phosphate,
substituent
at C-2
then leaves.
Ifproducing
the substituent
C-2
is a
3-H
be
by
and the
substituent
at at
C-2
then
leaves.
If the
substituent
at C-2
is ais
3-Hcan
can
beassisted
assisted
byphosphate,
phosphate,
substituent
C-2
then
leaves.
If the
substituent
at 3C-2
good
leaving
group (toluene
in the and
case the
of arylacetaldehydes),
1,3,5-trisubstituted
pyridiniums
will
good
leaving
group
(toluene
in in
thethe
case
ofofarylacetaldehydes),
1,3,5-trisubstituted
pyridiniums
33will
a good
leaving
group
(toluene
arylacetaldehydes),
1,3,5-trisubstituted
pyridiniums
will
predominantly
be formed
(Scheme
1).case
Otherwise,
the 1,2,3,5-tetrasubstituted
intermediate
will slowly
predominantly
be
(Scheme
the
intermediate
will slowly
predominantly
beformed
formed
(Scheme1).
1).Otherwise,
Otherwise,
the1,2,3,5-tetrasubstituted
1,2,3,5-tetrasubstituted
intermediate
oxidise
to generate
the corresponding
1,2,3,5-tetrasubstituted
pyridinium Chichibabin
saltwill
1. slowly
oxidise
totogenerate
the
1,2,3,5-tetrasubstituted pyridinium
Chichibabin
salt
oxidise
generate
thecorresponding
corresponding
Chichibabin
salt1.1. of new
There
is a growing
need for new1,2,3,5-tetrasubstituted
antibiotics and over pyridinium
the last 30 years
the number
There
need
for
new
and
the
the number
ofofnew
Thereis
isaagrowing
growing
need
for
newantibiotics
antibiotics
andover
over
thelast
last30
30years
years
new
antibiotics
entering
the clinic
has
decreased
significantly.
During
that
time
there the
has number
been a constant
antibiotics
entering
the
clinic
has
decreased
significantly.
During
that
time
there
has
been
a
constant
antibiotics
entering
the
clinic
has
decreased
significantly.
During
that
time
there
has
been
a
constant
rise in the resistance of pathogenic bacteria to the antibiotics in use. One of the major pathogens in
rise
resistance
ofofpathogenic
bacteria
antibiotics
use.
of the
inin
riseininthe
theand
resistance
pathogenic
bacteriatotothe
antibioticsininaureus.
use.One
One
themajor
majorpathogens
pathogens
hospitals
increasingly
in the community
isthe
Staphylococcus
Thisofbacterium
has
acquired
hospitals
and
increasingly
in
the
community
is
Staphylococcus
aureus.
This
bacterium
has
acquired
hospitals and
in the
is Staphylococcus
bacteriumInhas
resistance
overincreasingly
the last 25 years
tocommunity
methicillin and
in the last 15 aureus.
years toThis
vancomycin.
theacquired
light of
resistance
over
the
last
25
years
totomethicillin
and
ininthe
last
15
years
to
vancomycin.
InInthe
light
ofof
resistance
over
the
last
25
years
methicillin
and
the
last
15
years
to
vancomycin.
the
lightfor
this need for new antibiotics to treat S. aureus infections, and the anti-microbial activities reported
this
new
antibiotics
tototreat
infections,
anti-microbial
activities
reported for
thisneed
needfor
for[3],
new
antibiotics
treatS.S.aureus
aureus
infections,and
andthe
the
anti-microbial
activities
for
ficuseptine
selected
1,3,5-pyridinium
compounds
were
tested
for their ability
to reported
inhibit the
ficuseptine
[3],
selected
1,3,5-pyridinium
compounds
were
tested
for
their
ability
to
the
ficuseptine
selected
1,3,5-pyridinium
for their
ability concentration
to inhibitinhibit
the growth
growth
of S.[3],
aureus.
Agar
plate diffusioncompounds
assays and were
liquidtested
minimal
inhibitory
(MIC)
growth
of S. aureus.
Agar plate diffusion
assays
and
liquid minimal
concentration
(MIC)
of S. were
aureus.
Agar plate
assays
and
liquid
minimal
inhibitoryinhibitory
concentration
(MIC) tests
were
tests
carried
out todiffusion
assess the
potency
of
the compounds.
tests
were
carried
out
to
assess
the
potency
of
the
compounds.
carried
outexamples
to assess the
potency
the compounds.
Some
of the
agar of
plate
diffusion assays against a kanamycin control are shown in
Some examples of the agar plate diffusion assays against a kanamycin control are shown in
Figure 2 and the MIC data in Table 4. The data demonstrated the potency of some of the compounds
Figure 2 and the MIC data in Table 4. The data demonstrated the potency of some of the compounds
compared to the antibiotic kanamycin.
compared to the antibiotic kanamycin.
Molecules 2017, 22, 626
6 of 13
Some examples of the agar plate diffusion assays against a kanamycin control are shown in
Figure 2 and the MIC data in Table 4. The data demonstrated the potency of some of the compounds
compared
to the
antibiotic kanamycin.
Molecules 2017,
22, 426
6 of 13
OH
N
OH
3i
3c
N
Kanamycin control
N
OMe
3h
3q
N
N
OH
MeO
3j
OH
N
OH
5
Figure
Examplesofofthe
theagar
agarplate
platediffusion
diffusion assays
assays against
3 3
Figure
2. 2.
Examples
against S.
S.aureus
aureuswith
withselected
selectedpyridiniums
pyridiniums
(at
400
μg/mL)
and
the
kanamycin
control
(at
100
μg/mL).
(at 400 µg/mL) and the kanamycin control (at 100 µg/mL).
Table 4. MIC data for a range of the pyridiniums 3 determined from MIC tests.
Table 4. MIC data for a range of the pyridiniums 3 determined from MIC tests.
Compound
Compound
3a
3b3a
3c3b
3d3c
3e3d
3e
3f
3f
3g3g
1
1
MIC (μg/mL)
MICn.d.
(µg/mL)
32
n.d.
32
n.d.
n.d.
64
64
16
16
64
64
16
16
Compound
Compound
3h
3h3i
3i3j
3j3k
3k
3o
3o
3q
3q
3r3r
1
1
MIC (μg/mL)
MIC (µg/mL)
64
n.d.
64
n.d.64
64
n.d.
n.d.32
32
32
32
n.d.
n.d.
1
growth
inhibition
detected
(n.d.)
represents
compounds
with
MIC
256for
μg/mL.
MICagainst
for
1 NoNo
growth inhibition detected (n.d.) represents compounds with MIC > 256 µg/mL.>MIC
kanamycin
kanamycin
against
S. aureus
been
reported as 3.5 μg/mL [28].
S. aureus
has been
reported
as 3.5 has
µg/mL
[28].
From the compounds tested it was notable that the two most potent anti-bacterials against
From the compounds tested it was notable that the two most potent anti-bacterials against
S. aureus were compounds 3e and 3g (Figure 3). Both had an aromatic ring at C-3 and C-5 of the
S. aureus were compounds 3e and 3g (Figure 3). Both had an aromatic ring at C-3 and C-5 of the
pyridinium ring, indeed for 3r where an isopropyl group was present, no growth inhibitory
pyridinium ring, indeed for 3r where an isopropyl group was present, no growth inhibitory properties
properties were noted. In addition, 3g had a 5-bromo-3-hydroxyphenyl group attached via an ethyl
were
noted. In addition, 3g had a 5-bromo-3-hydroxyphenyl group attached via an ethyl spacer to
spacer to N-1, whereas in 3e a 3-bromophenyl group was present. Overall this data suggests that
N-1,
whereas
in 3e a 3-bromophenyl
group
wasare
present.
Overall
suggests
aromatic
aromatic
substituents
at positions C-3
and C-5
preferred,
and this
that data
substitution
of that
the phenyl
substituents
at
positions
C-3
and
C-5
are
preferred,
and
that
substitution
of
the
phenyl
rings
is
tolerated,
rings is tolerated, as demonstrated by 3o and 3q. Also, that substitution of the N-1 phenethyl group
by a halogen leads to good inhibitory growth properties. However, it is not essential for an aromatic
group to be present at N-1, for example as with 3j, for inhibitory growth effects to be conferred.
Molecules 2017, 22, 626
7 of 13
as demonstrated by 3o and 3q. Also, that substitution of the N-1 phenethyl group by a halogen leads
to good inhibitory growth properties. However, it is not essential for an aromatic group to be present
at N-1, for
example
Molecules
2017,
22, 426 as with 3j, for inhibitory growth effects to be conferred.
7 of 13
Figure
Figure 3.
3. Structures
Structures of
of 3e
3e and
and 3g.
3g.
These results point to compounds that can now be made to explore the structure activity
These results point to compounds that can now be made to explore the structure activity
relationship of the various substituents that can be placed on the C-1, C-3, and C-5 positions of the
relationship of the various substituents that can be placed on the C-1, C-3, and C-5 positions of
pyridine ring. The activity against other bacteria will be reported elsewhere.
the pyridine ring. The activity against other bacteria will be reported elsewhere.
4. Experimental Section
3. Experimental Section
4.1.
3.1. General
General Information
Information and
and Methods
Methods
4.1.1.
3.1.1. Chemistry
All reagents were obtained from commercial sources and used as received unless otherwise
stated.
254
precoated
plastic
plates
and
compounds
visualised
by
stated. TLC
TLCwas
wasperformed
performedon
onKieselgel
Kieselgel6060FF
precoated
plastic
plates
and
compounds
visualised
254
exposure
to UV
light,
potassium
permanganate,
phosphomolybdic
acid (PMA)
or ninhydrin.
Flash
by exposure
to UV
light,
potassium
permanganate,
phosphomolybdic
acid (PMA)
or ninhydrin.
column
chromatography
was carried
out using
(particule
size 40–63
HPLC
Flash column
chromatography
was carried
out silica
usinggel
silica
gel (particule
size μm).
40–63Preparative
µm). Preparative
were
on a Prostar
instrument
(Varian
Inc., Middelburg,
The Netherlands)
equipped
with
HPLCperformed
were performed
on a Prostar
instrument
(Varian
Inc., Middelburg,
The Netherlands)
equipped
an
autosampler,
a UV-visible
detector
and aand
DiscoveryBIO
wide wide
Pore C18-10
Supelco
column
(25 ×
with
an autosampler,
a UV-visible
detector
a DiscoveryBIO
Pore C18-10
Supelco
column
2.12
were monitored
at 280 nmatand
out carried
using a out
gradient
to 90% acetonitrile
(25 ×cm).
2.12Elutions
cm). Elutions
were monitored
280carried
nm and
usingofa5 gradient
of 5% to
13C-NMR spectra
13 C-NMR
against
water (+0.1%
trifluoroacetic
acid).
NMR: 1H- and
were
recorded
at 298were
K at
90% acetonitrile
against
water (+0.1%
trifluoroacetic
acid).
NMR: 1 H- and
spectra
the
field indicated
using
Avance
500 andusing
Avance
III 600
(Bruker,
(UK) Ltd, Coventry,
recorded
at 298 K at
the field
indicated
Avance
500spectrometers.
and Avance III
600 spectrometers.
(Bruker,
UK).
constants
are measured
in Hertz
(Hz)
and multiplicities
for 1H-NMR
couplings are
(UK) Coupling
Ltd., Coventry,
UK).(J)Coupling
constants
(J) are
measured
in Hertz (Hz)
and multiplicities
for
1
shown
as couplings
s (singlet), are
d (doublet),
hept
(heptet) and
m (multiplet).
Chemical
(in ppm)
H-NMR
shown ast (triplet),
s (singlet),
d (doublet),
t (triplet),
hept (heptet)
andshifts
m (multiplet).
are
given shifts
relative
to tetramethylsilane
referenced to residual
protonated
solvent.
Infrared
Chemical
(in ppm)
are given relativeand
to tetramethylsilane
and referenced
to residual
protonated
spectra
were
recorded
on
a
Spectrum
100
FTIR
spectrometer
(Perkin
Elmer,
Shelton,
CT,
USA).
Mass
solvent. Infrared spectra were recorded on a Spectrum 100 FTIR spectrometer (Perkin Elmer, Shelton,
spectrometry
analyses
were performed
at theperformed
UCL Chemistry
MassChemistry
Spectrometry
Facility
using a
CT, USA). Mass
spectrometry
analyses were
at the UCL
Mass
Spectrometry
Finnigan
MATa900
XP andMAT
MicroMass
LC mass Quattro
spectrometer
(Waters
UK, Elstree,
UK). TFA
Facility using
Finnigan
900 XPQuattro
and MicroMass
LC mass
spectrometer
(Waters
UK,
− salt and NMR signals
13C-NMR data.
−
refers
to
the
CF
3
CO
2
are
not
recorded
for
TFA
in
the
Elstree, UK). TFA refers to the CF3 CO2 salt and NMR signals are not recorded for TFA in the
13 C-NMR data.
4.1.2. Biological Assays
3.1.2. Biological Assays
Plate zone assays
Plate Zone Assays
Staphylococcus aureus was grown overnight in LB broth (Oxoid Ltd, Basingstoke, UK) and 100 μL
Staphylococcus
aureus
wasofgrown
overnight
in LBabroth
(Oxoid
Ltd., A
Basingstoke,
and 100
was spread
onto the
surface
LB agar
plates using
sterile
spreader.
sterile glassUK)
pipette
withµL
a
was
spread
onto
the
surface
of
LB
agar
plates
using
a
sterile
spreader.
A
sterile
glass
pipette
with
diameter of 6 mm was used to punch out wells and the agar removed from the wells. 50 μL of
a diameter of
was used to
out wells
and
the agar
from the(positive
wells. 50control
µL of
compound,
at 6a mm
concentration
of punch
400 μg/mL
in RO
water,
or a removed
control solution
compound,atat100
a concentration
of 400
µg/mL
inDMSO/water)
RO water, or was
a control
solution
(positive
control
kanamycin
μg/mL; negative
control
1:12.5
pipetted
into each
well. The
LB
kanamycin
at
100
µg/mL;
negative
control
1:12.5
DMSO/water)
was
pipetted
into
each
well.
The
agar plates were then left to incubate at 37 °C for 18–24 h. The zone diameters were measured at 18LB
h.
agar plates were then left to incubate at 37 ◦ C for 18–24 h. The zone diameters were measured at 18 h.
Minimimal Inhibitory Concentration (MIC)
Fifty mL of LB broth was inoculated with a 1 mL overnight culture of Staphylococcus aureus, then
450 μL of the inoculated broth was pipetted into each well of a 96 deep square well plate. 50 μL of
compound was pipetted into each well; the compound was taken from the 256 μg/mL to 2 μg/mL
dilution series. A 5 mg/mL stock concentration of the test compounds was diluted with Reverse
Molecules 2017, 22, 626
8 of 13
Minimimal Inhibitory Concentration (MIC)
Fifty mL of LB broth was inoculated with a 1 mL overnight culture of Staphylococcus aureus,
then 450 µL of the inoculated broth was pipetted into each well of a 96 deep square well plate.
50 µL of compound was pipetted into each well; the compound was taken from the 256 µg/mL to
2 µg/mL dilution series. A 5 mg/mL stock concentration of the test compounds was diluted with
Reverse Osmosis (RO) water in clean, sterile 1.5 mL Eppendorf Micro-centrifuge tubes to a starting
concentration of 256 µg/mL. A 2-fold dilution series was made using RO water down to a concentration
2 µg/mL. There were 8 different dilutions in each series ranging from 256 µg/mL to 2 µg/mL.
After growth of the plate at 37 ◦ C for 18–24 h with shaking, the wells with no growth were recorded as
the MIC.
3.2. Procedure A for Synthesis of the 1,3,5-Substituted Pyridinium Salts
Amine (1 equiv.) and aldehyde (5 equiv.) were dissolved in a 1:1 mixture of methanol/phosphate
buffer (10 mL, 0.25 M solution at pH 6). The resulting solution was stirred at 100 ◦ C for 12 h. The crude
mixture was cooled to r.t. and purified by preparative HPLC (Gradient 1). Fractions containing the
desired product were combined, concentrated under vacuum and co-evaporated with methanol
(3 × 20 mL).
1-(4-Hydroxyphenethyl)-3,5-diphenylpyridinium·TFA (3a·TFA). Compound 3a was prepared according to
procedure A from tyramine·HCl (87 mg, 0.50 mmol) and phenylacetaldehyde (300 mg, 2.50 mmol).
The crude product was purified by preparative HPLC (retention time 15.5 min) to give 3a·TFA as
a pale yellow oil (162 mg, 70%). νmax (neat)/cm−1 3064, 1671, 1613, 1596, 1517; 1 H-NMR (500 MHz;
CD3 OD) δ 3.29–3.32 (2H, m, CH2 CH2 N+ ), 4.95 (2H, t, J = 6.7 Hz, CH2 N+ ), 6.74 (2H, d, J = 8.5 Hz, 300 -H,
500 -H), 6.98 (2H, d, J = 8.5 Hz, 200 -H, 600 -H), 7.56–7.60 (6H, m, Ph), 7.72–7.76 (4H, m, Ph), 8.91 (2H, d,
J = 1.7 Hz, 2-H, 6-H), 8.95 (1H, t, J = 1.7 Hz, 4-H); 13 C-NMR (125 MHz; CD3 OD) δ 37.5, 64.6, 116.7,
127.2, 128.5, 130.6, 131.1, 131.3, 134.5, 141.4, 141.7, 142.5, 158.1; m/z [HRMS ES+] found [M − TFA]+
352.1700. C25 H22 NO+ requires 352.1701.
1-Phenethyl-3,5-diphenylpyridinium·TFA (3b·TFA). Compound 3b was prepared according to procedure
A from phenethylamine (61 mg, 0.50 mmol) and phenylacetaldehyde (300 mg, 2.50 mmol). The crude
product was purified by preparative HPLC (retention time 16.5 min) to give 3b·TFA as a pale yellow
oil (121 mg, 54%). νmax (neat)/cm−1 3064, 1683, 1597, 1482; 1 H-NMR (500 MHz; CD3 OD) δ 3.50 (2H, t,
J = 6.9 Hz, CH2 CH2 N+ ), 5.10 (2H, t, J = 6.9 Hz, CH2 N+ ), 7.28 (2H, d, J = 6.8 Hz, 200 -H, 600 -H), 7.33–7.41
(3H, m, Ph), 7.64–7.66 (6H, m, Ph), 7.80–7.83 (4H, m, Ph), 9.05 (1H, t, J = 1.6 Hz, 4-H), 9.07 (2H, d,
J = 1.6 Hz, 2-H, 6-H); 13 C-NMR (125 MHz; CD3 OD) δ 38.2, 64.2, 117.9, 128.5, 130.0, 130.5, 131.4, 134.5,
136.9, 141.6, 141.7, 142.6; m/z [HRMS ES+] found [M − TFA]+ 336.1750. C25 H22 N+ requires 336.1752.
3,5-Diphenyl-1-[2-(pyridin-2-yl)ethyl]pyridinium·TFA (3c·TFA). Compound 3c was prepared according
to procedure A from 2-(2-aminoethyl)pyridine (35 mg, 0.29 mmol) and phenylacetaldehyde (170 mg,
1.4 mmol). The crude product was purified by preparative HPLC (retention time 13.5 min) to give
3c·TFA as a pale yellow oil (68 mg, 52%). νmax (neat)/cm−1 3077, 1635, 1598, 1483; 1 H-NMR (500 MHz;
CD3 OD) δ 3.85 (2H, t, J = 7.4 Hz, CH2 CH2 N+ ), 5.21 (2H, t, J = 7.4 Hz, CH2 N+ ), 7.57–7.64 (6H, m, Ph),
7.73 (1H, dd, J = 7.6 and 4.9 Hz, 500 -H), 7.83 (1H, d, J = 7.6 Hz, 300 -H), 7.87–7.89 (4H, m, Ph), 8.27 (1H, t,
J = 7.6 Hz, 400 -H), 8.68 (1H, d, J = 4.9 Hz, 600 -H), 9.04 (1H, t, J = 1.5 Hz, 4-H), 9.27 (2H, d, J = 1.5 Hz, 2-H,
6-H); 13 C-NMR (125 MHz; CD3 OD) δ 37.3, 61.6, 126.0, 127.8, 128.8, 129.5, 130.8, 131.6, 134.7, 142.2, 142.3,
143.2, 144.4, 146.6, 154.5; m/z [HRMS ES+] found [M − TFA]+ 337.1708. C24 H21 N2 + requires 337.1705.
3,5-Diphenyl-1-[2-(thiophen-2-yl)ethyl]pyridinium·TFA (3d·TFA). Compound 3d was prepared according
to procedure A from thiophene-2-ethylamine (36 mg, 0.28 mmol) and phenylacetaldehyde (170 mg,
1.4 mmol). The crude product was purified by preparative HPLC (retention time 16.2 min) to give
3d·TFA as a pale yellow oil (48 mg, 38%). νmax (neat)/cm−1 3065, 2940, 1680, 1597, 1483; 1 H-NMR
(500 MHz; CD3 OD) δ 3.67 (2H, t, J = 6.6 Hz, CH2 CH2 N+ ), 5.02 (2H, t, J = 6.6 Hz, CH2 N+ ), 6.87 (1H, m,
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300 -H), 6.96 (1H, dd, J = 5.1 and 3.5 Hz, 400 -H), 7.33 (1H, dd, J = 5.1 and 1.1 Hz, 500 -H), 7.56–7.62
(6H, m, Ph), 7.77–7.79 (4H, m, Ph), 8.98 (1H, t, J = 1.7 Hz, 4-H), 9.04 (2H, d, J = 1.7 Hz, 2-H, 6-H);
13 C-NMR (125 MHz; CD OD) δ 32.2, 64.4, 126.7, 128.5, 128.7, 128.8, 130.8, 131.6, 134.7, 138.5, 141.9,
3
142.0, 142.9; m/z [HRMS ES+] found [M − TFA]+ 342.1302. C23 H20 NS+ requires 342.1316.
1-(2-[3-Bromophenethyl])-3,5-diphenylpyridinium·TFA (3e·TFA). Compound 2e was prepared according
to procedure A from 3-bromophenethylamine (100 mg, 0.50 mmol) and phenylacetaldehyde (300 mg,
2.5 mmol). The crude product was purified by preparative HPLC (retention time 17.2 min) to give
3e·TFA as a pale yellow oil (131 mg, 50%). νmax (neat)/cm−1 3086, 2979, 1685, 1601, 1488; 1 H-NMR
(500 MHz; CD3 OD) δ 3.42 (2H, t, J = 7.2 Hz, CH2 CH2 N+ ), 5.00 (2H, t, J = 7.2 Hz, CH2 N+ ), 7.21–7.27
(2H, m, 200 -H, 500 -H), 7.42–7.47 (2H, m, 400 -H, 600 -H), 7.57–7.61 (6H, m, Ph), 7.79 (4H, d, J = 6.3 Hz,
2 × 20 -H, 60 -H), 8.97 (1H, t, J = 1.7 Hz, 4-H), 9.09 (2H, d, J = 1.7 Hz, 2-H, 6-H); 13 C-NMR (125 MHz;
CD3 OD) δ 37.8, 63.9, 124.0, 128.8, 129.1, 130.8, 131.6, 131.8, 131.9, 133.3, 134.7, 139.8, 141.9, 142.0, 143.0;
m/z [HRMS ES+] found [M − TFA]+ 414.0840. C25 H21 79 BrN+ requires 414.0857.
1-[2-(3-Nitrophenethyl)]-3,5-diphenylpyridinium·TFA (3f·TFA). Compound 3f was prepared according
to procedure A from 3-nitrophenethylamine (17 mg, 0.10 mmol) and phenylacetaldehyde (60 mg,
0.5 mmol). The crude product was purified by preparative HPLC (retention time 16.0 min) to give
3f·TFA as a pale yellow oil (22 mg, 45%). νmax (neat)/cm−1 3070, 1655, 1598, 1561; 1 H-NMR (500 MHz;
CD3 OD) δ 3.59 (2H, t, J = 7.4 Hz, CH2 CH2 N+ ), 5.04 (2H, t, J = 7.4 Hz, CH2 N+ ), 7.59–7.64 (7H, m, 500 -H
and Ph), 7.71 (1H, d, J = 7.6 Hz, 600 -H), 7.83 (4H, dd, J = 7.8 and 1.7 Hz, 2 × 20 -H, 60 -H), 8.16 (1H, d,
J = 8.2 Hz, 400 -H), 8.20 (1H, s, 200 -H), 9.04 (1H, t, J = 1.6 Hz, 4-H), 9.21 (2H, d, J = 1.6 Hz, 2-H, 6-H);
13 C-NMR (125 MHz; CD OD) δ 37.7, 63.6, 123.6, 125.1, 128.8, 130.8, 131.4, 131.6, 134.7, 136.6, 139.5,
3
142.0, 142.1, 143.1, 149.5; m/z [HRMS ES+] found [M − TFA]+ 381.1604. C25 H21 N2 O2 + requires
381.1603.
3-(2-Aminoethyl)-4-bromophenol (6g). The reaction was carried out under anhydrous conditions.
To a solution of 2-(2-bromo-5-methoxyphenyl)ethylamine [29] (120 mg, 0.52 mmol) in dichloromethane
(20 mL) at −78 ◦ C, boron tribromide (1.3 mL, 1.3 mmol; 1 M solution in hexane) was added.
The mixture was warmed to r.t. and stirred for 20 h, then cooled to −78 ◦ C and water (50 mL)
added dropwise. The aqueous layer was extracted with dichloromethane (3 × 50 mL), filtered and
concentrated under reduced pressure to give 6g [30] as a colourless oil (110 mg, 97%). 1 H-NMR (600
MHz; D2 O) δ 3.09 (2H, t, J = 7.5 Hz, CH2 CH2 N), 3.29 (2H, t, J = 7.5 Hz, CH2 N), 6.77 (1H, dd, J = 8.7 and
3.0 Hz, 6-H), 6.90 (1H, d, J = 3.0 Hz, 2-H), 7.52 (1H, d, J = 8.7 Hz, 5-H); 13 C-NMR (150 MHz; D2 O) δ 33.9,
39.7, 114.4, 117.1, 118.7, 134.6, 137.7, 156.1; m/z [HRMS ES+] found [MH]+ 216.0030. C8 H11 79 BrNO
requires 216.0024.
1-(2-Bromo-5-hydroxyphenethyl)-3,5-diphenylpyridinium·TFA (3g·TFA). Compound 6g (50 mg, 0.23 mmol)
and phenylacetaldehyde (33 mg, 0.28 mmol) in 10 mL of a 1:1 mixture of acetonitrile/phosphate
buffer (0.1 M solution at pH 6) were stirred at 60 ◦ C for 12 h. The crude product was purified
by preparative HPLC (retention time 16.5 min) and fractions containing the desired product were
combined, concentrated and co-evaporated with methanol (3 × 20 mL) to give 3g·TFA (16 mg, 13%)
as a pale yellow oil 1 H-NMR (600 MHz; CD3 OD) δ 3.51 (2H, t, J = 6.6 Hz, CH2 CH2 N+ ), 5.05 (2H, t,
J = 6.6 Hz, CH2 N+ ), 6.66–6.70 (2H, m, 400 -H, 600 -H), 7.37 (1H, d, J = 8.3 Hz, 300 -H), 7.57–7.62 (6H, m,
Ph), 7.74–7.77 (4H, m, Ph), 8.95 (2H, d, J = 1.6 Hz, 2-H, 6-H), 9.02 (1H, t, J = 1.6 Hz, 4-H); 13 C-NMR
(125 MHz; CD3 OD) δ 38.0, 63.3 114.2, 117.9, 119.3, 128.7, 130.8, 131.6, 134.7, 135.0, 137.4, 142.0, 142.2,
142.9, 159.1; m/z [HRMS EI+] found [M − TFA]+ 430.0815. C25 H21 79 BrNO+ requires 430.0807.
1-(3-Hydroxybenzyl)-3,5-diphenylpyridinium·TFA (3h·TFA). Compound 3h was prepared according
to procedure A, from 3-aminomethylphenol (62 mg, 0.50 mmol) and phenylacetaldehyde (300 mg,
2.50 mmol). The crude product was purified by preparative HPLC (retention time 14.4 min) to give
3h·TFA as a pale yellow oil (147 mg, 65%). νmax (neat)/cm−1 3070, 2927, 1661, 1586, 1482; 1 H- NMR
(500 MHz; CD3 OD) δ 5.88 (2H, s, CH2 N+ ), 6.88 (1H, d, J = 7.9 Hz, 600 -H), 6.97 (1H, s, 200 -H), 7.02 (1H, d,
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J = 7.9 Hz, 400 -H), 7.27 (1H, t, J = 7.9 Hz, 500 -H), 7.57–7.63 (6H, m, Ph), 7.84–7.88 (4H, m, Ph), 9.04 (1H, s,
4-H), 9.30 (2H, s, 2-H, 6-H); 13 C-NMR (125 MHz; CD3 OD) δ 66.0, 116.4, 117.9, 120.5, 128.8, 130.8, 131.6,
131.9, 134.7, 136.1, 141.9, 142.3, 143.3, 159.7; m/z [HRMS ES+] found [M − TFA]+ 338.1513. C24 H20 NO+
requires 338.1539.
1-(2,3-Dihydroxypropyl)-3,5-diphenylpyridinium·TFA (3i·TFA). Compound 3i was prepared according
to procedure A, from 3-aminopropane-1,2-diol (45 mg, 0.49 mmol) and phenylacetaldehyde (300
mg, 2.50 mmol). The crude product was purified by preparative HPLC (retention time 13.0 min)
to give 3i·TFA as a pale yellow oil (147 mg, 72%). νmax (neat)/cm−1 3292, 3070, 1667, 1597, 1485;
1 H-NMR (500 MHz; CD OD) δ 3.60 (1H, dd, J = 11.4 and 6.0 Hz, CHHOH), 3.76 (1H, dd, J = 11.4 and
3
4.7 Hz, CHHOH), 4.15–4.18 (1H, m, CHOH), 4.74 (1H, dd, J = 13.2 and 8.5 Hz, CHHN+ ), 4.97 (1H, dd,
J = 13.2 and 3.0 Hz, CHHN+ ), 7.57–7.64 (6H, m, Ph), 7.87–7.91 (4H, m, Ph), 9.03 (1H, t, J = 1.7 Hz, 4-H),
9.21 (2H, d, J = 1.7 Hz, 2-H, 6-H); 13 C-NMR (125 MHz; CD3 OD) δ 64.3, 65.7, 71.9, 128.8, 130.8, 131.5,
134.9, 141.8, 142.5, 142.9; m/z [HRMS EI] found [M − TFA]+ 306.1488. C20 H20 NO2 + requires 306.1489.
1-(6-Hydroxyhexyl)-3,5-diphenylpyridinium·TFA (3j·TFA). Compound 3j was prepared according to
procedure A, from 6-hydroxyhexylamine (59 mg, 0.50 mmol) and phenylacetaldehyde (300 mg,
2.50 mmol). The crude product was purified by preparative HPLC (retention time 15.3 min) to give
3j·TFA as a pale yellow oil (153 mg, 69%). νmax (neat)/cm−1 3070, 2932, 1598, 1484; 1 H-NMR
(500 MHz; CD3 OD) δ 1.49–1.85 (6H, m, 3 × CH2 ), 2.14–2.19 (2H, m, CH2 ), 3.67 (2H, t, J = 6.3 Hz,
CH2 OH), 4.75 (2H, t, J = 7.8 Hz, CH2 N+ ), 7.58–7.65 (6H, m, Ph), 7.88–7.92 (4H, m, Ph), 9.03 (1H, t, J =
1.6 Hz, 4-H), 9.29 (2H, d, J = 1.6 Hz, 2-H, 6-H); 13 C-NMR (125 MHz; CD3 OD) δ 26.2, 26.8, 28.9, 32.5,
62.6, 63.4, 128.8, 130.8, 131.6, 134.9, 141.8, 142.0, 143.2; m/z [HRMS ES+] found [M − TFA]+ 332.2018.
C23 H26 NO+ requires 332.2014.
1-(4-Carboxybutyl)-3,5-diphenylpyridinium·TFA (3k·TFA). Compound 3k was prepared according to
procedure A, from 4-aminobutyric acid (51 mg, 0.49 mmol) and phenylacetaldehyde (300 mg,
2.50 mmol). The crude product was purified by preparative HPLC (retention time 14.2 min) to give
3k·TFA as a pale yellow oil (134 mg, 63%). νmax (neat)/cm−1 3200, 2943, 1668, 1598, 1483; 1 H-NMR
(500 MHz; CD3 OD) δ 2.43 (2H, m, CH2 CH2 CO2 H), 2.56 (2H, t, J = 6.8 Hz, CH2 CO2 H), 4.82 (2H, t,
J = 7.3 Hz, CH2 N+ ), 7.56–7.64 (6H, m, Ph), 7.88–7.92 (4H, m, Ph), 9.00 (1H, t, J = 1.6 Hz, 4-H), 9.28 (2H, d,
J = 1.6 Hz, 2-H, 6-H); 13 C-NMR (125 MHz; CD3 OD) δ 27.6, 31.3, 62.7, 128.8, 130.8, 131.5, 134.9, 141.9,
142.2, 143.1, 175.7; m/z [HRMS ES+] found [M − TFA]+ 318.1485. C21 H20 NO2 + requires 318.1494.
1-Cyclohexyl-3,5-diphenylpyridinium·TFA (3l·TFA). Compound 3l was prepared according to procedure
A from cyclohexylamine (50 mg, 0.50 mmol) and phenylacetaldehyde (300 mg, 2.50 mmol). The crude
product was purified by preparative HPLC (retention time 16.2 min) to give 3l·TFA as a pale yellow
oil (113 mg, 53%). 1 H-NMR (500 MHz; CD3 OD) δ 1.46 (1H, app. qt, J = 13.2 and 3.6 Hz, 400 -Hax ),
1.63 (2H, app. q, J = 10.2 Hz, 2 × 200 -Hax ), 1.82 (1H, br d, J = 13.2 Hz, 400 -Heq ), 2.05 (2H, br d,
J = 13.8 Hz, 2 × 200 -Heq ), 2.18 (2H, qd, J = 12.3 and 3.6 Hz, 2 × 300 -Hax ), 2.32 (2H, br d, J = 10.6 Hz,
2 × 300 -Heq ), 4.82 (1H, m, CHN+ ), 7.56–7.64 (6H, m, Ph), 7.89–7.92 (4H, m, Ph), 9.00 (1H, d, J = 1.6 Hz,
4-H), 9.25 (2H, d, J = 1.6 Hz, 2-H, 6-H); 13 C-NMR (125 MHz; CD3 OD) δ 25.5, 26.6, 34.3, 74.3, 129.0,
130.8, 131.5, 135.0, 140.5, 142.1, 143.3; m/z (ES+) 314 (M+ , 100%), 232 (12); m/z [HRMS ES+] found
[M − TFA]+ 314.1906. C23 H24 N+ requires 314.1909.
3,5-Bis-(4-hydroxyphenyl)-1-(4-hydroxyphenethyl)pyridinium·TFA (3o·TFA). Compound 3o was prepared
according to procedure A, from tyramine·HCl (5.0 mg, 0.028 mmol) and 4-hydroxyphenylacetaldehyde [22] (17 mg, 0.13 mmol). The crude product was purified by preparative HPLC
(retention time 13.5 min) to give 3o·TFA as a pale yellow oil (9.2 mg, 66%). 1 H-NMR (600 MHz;
CD3 OD) δ 3.28 (2H, t, J = 6.7 Hz, CH2 CH2 N+ ), 4.87 (2H, t, J = 6.7 Hz, CH2 N+ ), 6.73 (2H, d, J = 8.2 Hz,
300 -H, 500 -H), 6.94–6.98 (6H, m, 2 × (30 H, 50 -H), 200 -H, 600 -H), 7.57 (d, J = 8.2 Hz, 2 × 20 H, 60 -H),
8.71 (2H, d, J = 1.6 Hz, 2-H, 6-H), 8.77 (1H, t, J = 1.6 Hz, 4-H); 13 C-NMR (150 MHz; CD3 OD) δ 37.7,
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64.6, 116.9, 117.5, 125.6, 127.5, 130.1, 131.3, 139.2, 139.9, 142.5, 158.3, 161.2; m/z [HRMS ES+] found
[M − TFA]+ 384.1602. C25 H22 NO3 + requires 384.1600.
1-(4-Hydroxyphenethyl)-3,5-di(thiophen-2-yl)pyridinium·TFA (3p·TFA). Compound 2p was prepared
according to procedure A, from tyramine·HCl (5.0 mg, 0.028 mmol) and thiophen-2-ylacetaldehyde [22] (17 mg, 0.14 mmol). The crude product was purified by preparative HPLC
(retention time 13.5 min) to give 3p·TFA as a pale yellow oil (5.6 mg, 42%). 1 H-NMR (600 MHz; CD3 OD)
δ 3.29 (2H, t, J = 6.8 Hz, CH2 CH2 N+ ), 4.87 (2H, t, J = 6.8 Hz, CH2 N+ ), 6.73 (2H, d, J = 8.5 Hz, 300 -H, 500 -H),
6.99 (2H, d, J = 8.5 Hz, 200 -H, 600 -H), 7.27 (2H, dd, J = 4.9 and 3.8 Hz, 40 -H), 7.75–7.78 (4H, m, 2 × 300 -H,
500 -H), 8.79 (1H, t, J = 1.7 Hz, 4-H), 8.83 (2H, d, J = 1.7 Hz, 2-H, 6-H); 13 C-NMR (150 MHz; CD3 OD)
δ 37.5, 64.8, 116.9, 127.3, 129.7, 130.3, 131.2, 131.3, 136.4, 136.7, 136.9, 139.7, 158.3; m/z [HRMS ES+]
found [M − TFA]+ 364.0820. C21 H18 NOS2 + requires 364.0830.
1-(4-Hydroxyphenethyl)-3,5-bis-(4-methoxyphenyl)pyridinium·TFA (3q·TFA). Compound 3q was prepared
according to procedure A, from tyramine·HCl (10 mg, 0.056 mmol) and 4-methoxyphenylacetaldehyde [24] (38 mg, 0.25 mmol). The crude product was purified by preparative HPLC
(retention time 16.2 min) to give 3q·TFA as a pale yellow oil (17 mg, 58%). 1 H-NMR (600 MHz;
CD3 OD) δ 3.29 (2H, t, J = 6.7 Hz, CH2 CH2 N+ ), 3.88 (6H, s, 2 × OMe), 4.90 (2H, t, J = 6.7 Hz, CH2 N+ ),
6.74 (2H, d, J = 8.5 Hz, 300 -H, 500 -H), 6.97 (2H, d, J = 8.5 Hz, 200 -H, 600 -H), 7.11 (4H, d, J = 8.7 Hz, 30 -H,
50 -H), 7.68 (4H, d, J = 8.7 Hz, 20 -H, 60 -H), 8.78 (2H, d, J = 1.6 Hz, 2-H, 6-H), 8.83 (1H, t, J = 1.6 Hz, 4-H);
13 C- NMR (150 MHz; CD OD) δ 37.7, 56.0, 64.7, 116.1, 116.9, 126.9, 127.5, 130.3, 131.3, 139.8, 140.4,
3
142.3, 158.3, 163.1; m/z [HRMS ES+] found [M − TFA]+ 412.1909. C27 H26 NO3 + requires 412.1913.
1-(4-Hydroxyphenethyl)-3,5-di(isopropyl)pyridinium·TFA and 1-(4-Hydroxyphenethyl)-2-isobutyl-3,5-di(isopropyl)
pyridinium·TFA (3r·TFA and 1r·TFA). Compounds 3r and 1r were prepared according to procedure
A, from tyramine HCl (52 mg, 0.30 mmol) and isovaleraldehyde (120 mg, 1.4 mmol). The crude
products were purified by preparative HPLC (retention times 15.3 min (3r) and 16.2 min (1r)) to give
3r·TFA (6 mg, 5%) and 1r·TFA (13 mg, 10%) as pale yellow oils. Compound 3r: 1 H-NMR (500 MHz;
CD3 OD) δ 1.28 (12H, d, J = 6.7 Hz, 2 × CH(CH3 )2 ), 3.05–3.17 (4H, m, CH(CH3 )2 , CH2 CH2 N+ ),
4.77 (2H, t, J = 6.6 Hz, CH2 N+ ), 6.65 (2H, d, J = 8.4 Hz, 30 -H, 50 -H), 6.79 (2H, d, J = 8.4 Hz, 20 -H, 60 -H),
8.29 (2H, s, 2-H, 6-H), 8.34 (1H, s, 4-H); 13 C-NMR (125 MHz; CD3 OD) δ 23.2, 33.2, 37.6, 64.1, 116.8,
127.2, 130.9, 141.7, 143.2, 150.7, 158.2; m/z [HRMS ES+] found [M − TFA]+ 284.2019. C19 H26 NO+
requires 284.2014. Compound 1r: 1 H-NMR (500 MHz; CD3 OD) δ 1.12 (6H, d, J = 6.7 Hz, CH(CH3 )2 ),
1.20 (6H, d, J = 6.9 Hz, CH(CH3 )2 ), 1.31 (6H, d, J = 6.8 Hz, CH(CH3 )2 ), 1.97–2.03 (1H, m, CH(CH3 )2 ),
2.92 (2H, d, J = 7.5 Hz, 2 × CH2 CH(CH3 )2 ), 2.98 (1H, hept, J = 6.9 Hz, CH(CH3 )2 ), 3.15 (2H, t, J = 6.4 Hz,
CH2 CH2 N+ ), 3.32–3.36 (1H, m, CH(CH3 )2 ), 4.86 (2H, t, J = 6.4 Hz, CH2 N+ ), 6.65 (2H, d, J = 8.5 Hz,
30 -H, 50 -H), 6.74 (2H, d, J = 8.5 Hz, 20 -H, 60 -H), 8.14 (1H, s, 6-H), 8.30 (1H, s, 4-H); 13 C-NMR (125 MHz;
CD3 OD) δ 22.2, 23.0, 23.5, 31.0, 31.5, 32.8, 36.8, 37.0, 61.7, 116.8, 127.1, 131.1, 142.7, 143.3, 147.7, 150.6,
153.7, 158.3; m/z [HRMS ES+] found [M − TFA]+ 340.2650. C23 H34 NO+ requires 340.2640.
4. Conclusions
New reaction conditions have been developed that promote the condensation of arylacetaldehydes
with a variety of amines to yield 1,3,5-pyridinium salts 3. The reaction is promoted by phosphate and
can be carried out under conditions suitable for intrinsically unstable substrates such as arylaldehydes:
the reaction is derived from the Chichibabin pyridine synthesis. These results could underpin the
involvement of phosphate-containing molecules (e.g., inorganic phosphate, DNA, ATP) as catalysts
for the production of many alkaloids in vivo. The evaluation of a selection of 1,3,5-trisubstituted
pyridinium salts as bacteriocides prompted the discovery of novel S. aureus inhibitors in the low
µg/mL range. These preliminary results may pave the way towards the discovery of new antibiotics
based on the yet unexploited 1,3,5-trisubstituted pyridinium scaffold.
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Acknowledgments: We thank the Biotechnology & Biological Sciences Research Council (BBSRC) UK
(BB/G014426/1) for funding T.P. and M.C.G., and the Nuffield Foundation summer placement student scheme for
funding M.E. We also thank Vishal Sanchaina and Pedro Lebre for help with the MIC assay.
Author Contributions: All authors conceived and designed the experiments; T.P. and M.C.G. performed the
experiments; M.E. helped perform the MIC experiments; H.C.H. and J.M.W. conceived the project and directed
the experiments; All authors analyzed the data; The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuscript.
Conflicts of Interest: The founding sponsors had no role in the design of the study; in the collection, analyses,
or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.
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Sample Availability: Samples of compounds 3 are available from the authors upon reasonable requests (where
materials are available).
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