Synthesis and Evaluation of a Series of Bis(pentylpyridinium) Compounds as Antifungal Agents

Article abstract of DOI:10.1002/cmdc.201800331

A series of bis(4-pentylpyridinium) compounds with a variety of spacers between the pyridinium headgroups was synthesised, and the antifungal activity of these compounds was investigated. Lengthening the alkyl spacer between the pentylpyridinium headgroups from 12 to 16 methylene units resulted in increased antifungal activity against C. neoformans and C. albicans, but also resulted in increased hemolytic activity and cytotoxicity against mammalian cells. However, inclusion of an ortho-substituted benzene ring in the centre of the alkyl spacer resulted in decreased cytotoxicity and hemolytic activity, while maintaining antifungal potency. Replacement of the alkyl and aromatic-containing spacers by more hydrophilic ethylene glycol groups resulted in a loss of antifungal activity. Some of the compounds inhibited fungal PLB1 activity, but the low correlation of this inhibition with antifungal potency indicates PLB1 inhibition is unlikely to be the predominant mode of antifungal action of this class of compounds, with preliminary studies suggesting they may act via disruption of fungal mitochondrial function.

Full text of DOI:10.1002/cmdc.201800331

Accepted Article
Title: Synthesis and Evaluation of a Series of Bis(pentylpyridinium)
Compounds as Antifungal Agents
Authors: Daniel Obando, Yasuko Koda, Namfon Pantarat, Sophie Lev,
Xiaoming Zuo, Johanes Bijosono Oei, Fred Widmer, Julianne
Djordjevic, Tania Sorrell, and Katrina Jolliffe
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To be cited as: ChemMedChem 10.1002/cmdc.201800331
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Synthesis and Evaluation of a Series of Bis(pentylpyridinium)
Compounds as Antifungal Agents
Daniel Obando,#, [a] Yasuko Koda,#, [a], [b] Namfon Pantarat,[b] Sophie Lev,[b] Xiaoming Zuo,[b] Johanes
Bijosono Oei,[b] Fred Widmer,[b] Julianne T. Djordjevic,[b] Tania C. Sorrell[b] and Katrina A. Jolliffe*[a]
[a]
Dr Daniel Obando, Dr Yasuko Koda, Professor K. A. Jolliffe (ORCID Katrina A. Jolliffe 0000-0003-1100-4544)
School of Chemistry
The University of Sydney
2006, NSW, Australia
E-mail: kate.jolliffe@sydney.edu.au
[b]
Dr Yasuko Koda, Ms Namfon Pantarat, Dr Sophie Lev, Dr Xiaoming Zuo, Mr Johanes Bijosons Oei, Dr Fred Widmer, Associate Professor Julianne T.
Djordjevic, Professor Tania C. Sorrell
Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute and the Centre for Clinical Research Excellence in Infections, Bioethics
and Haematological Malignancies
The University of Sydney
Westmead, 2145, NSW, Australia
These authors contributed equally
#
Supporting information for this article is given via a link at the end of the document.
Abstract: A series of bis(4-pentylpyridinium) compounds with a
variety of spacers between the pyridinium headgroups was
synthesised and the antifungal activity of these compounds has been
investigated. Lengthening the alkyl spacer between the
pentylpyridinium headgroups from twelve to sixteen methylene units
resulted in increased antifungal activity against C. neoformans and C.
albicans, but also resulted in increased hemolytic activity and
cytotoxicity against mammalian cells. However, inclusion of an ortho-
substituted benzene ring in the centre of the alkyl spacer resulted in
reduced cytotoxicity and hemolytic activity, while maintaining
antifungal potency. Replacement of the alkyl and aromatic-containing
spacers by more hydrophilic ethylene glycol groups resulted in a loss
of antifungal activity. Some of the compounds inhibited fungal PLB1
activity, but the low correlation of this inhibition with antifungal potency
indicates PLB1 inhibition is unlikely to be the predominant mode of
antifungal action of this class of compounds, with preliminary studies
that new antifungal agents with different modes of action to those
of currently available drugs are required. Given the significant
burden of infection in the developing world, it is imperative that
the search for such drugs should include compounds with
characteristics appropriate for use in resource-poor, as well as
resource-rich countries. These include ease of manufacture,
potent antifungal activity, an excellent safety profile and low cost.
Fungal virulence factors such as phospholipase B (PLB) are
potential targets for the development of new antifungal drugs. PLB
is a proven virulence determinant of the pathogenic fungi,
Candida albicans and Cryptococcus neoformans,5-9 and is also
secreted by other pathogenic fungi including Aspergillus spp.10
Cryptococcal PLB has been well characterised11 and a number of
biscationic compounds with antifungal activity have been found to
inhibit PLB activity to some extent. For example, a series of
bisquaternary ammonium salts with a spacer twelve methylene
suggesting they may act via disruption of fungal mitochondrial function. units in length between the positively charged head groups exhibit
antifungal activity with MICs of 1 to 2.5 µg/mL against C.
neoformans and C. albicans and for this series of compounds,
antifungal activity correlated with inhibition of cryptococcal PLB1
Introduction
activity.12 In contrast, a series of bis(aminopyridinium)alkanes was
found to exhibit good antifungal activity, but these compounds did
not inhibit cryptoccocal PLB. However, this second class of
compounds was significantly less toxic towards human
erythrocytes than the bis(quaternary ammonium) class.13
Combining the structural features of these two classes of
The incidence of life-threatening invasive fungal infections
(mycoses) has increased significantly over the past two decades.1
These are associated with high morbidity and mortality and a
significant global economic burden. While Candida and
Aspergillus infections are historically the major causes of
mycoses, Cryptococcus is the commonest cause of fungal
meningitis, affecting in particular, HIV-infected patients in
developing countries; globally, cryptococcal meningitis is
responsible for 15% of AIDS-related deaths.2 The number of
currently available drugs for treatment of invasive fungal
infections is limited and limitations of marketed drugs include an
incomplete spectrum of activity against pathogenic fungi,
toxicities, poor bioavailability, poor solubility or stability, and/or
high cost. In addition, resistance has emerged to commonly used
treatments (e.g. fluconazole)3 and there are currently no effective
drugs against emerging pathogens such as some Fusarium
species and Lomentospora prolificans.4 It is widely recognised
compounds led to the investigation of
a
series of
bis(alkylpyridinium)alkanes in which positively charged
alkylpyridinium headgroups were separated by a 12 carbon
alkane spacer. These compounds exhibited useful antifungal
activities against a spectrum of common and more resistant
emerging fungal pathogens and more favourable cytotoxicity
profiles than the bis(quaternary ammonium) series.14,15 While the
antifungal activity of this class of compounds may be partially
attributable to the inhibition of cryptococcal PLB, this is unlikely to
be their major mode of action.
Previous studies of the antifungal activities of these biscationic
compounds have focused on changes to the structure of the
biscationic headgroups, rather than the linker between them. We
1
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10.1002/cmdc.201800331
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now report the antifungal activity of
a
series of bis(4-
In order to investigate the effect of the incorporation of rigidity into
the linker between the headgroups, compounds 9-13 were
prepared, in which either one or two multiple bonds were included
in the spacer (Figure 1). Compounds 14-29 (Figure 2) were also
prepared in which aromatic groups were incorporated into the
spacer units. Two series of these compounds were prepared; one
in which alkyl groups were appended directly to the aromatic
spacer, together with a second series of analogues in which the
inclusion of two alkynes conjugated to the aromatic ring provided
spacers with a greater degree of rigidity.
pentylpyridinium) compounds in which the two cationic
headgroups are held constant while the linker between these is
modified. Modification of the linker in such compounds provides a
wide scope to allow optimisation of the cytotoxicity and
pharmacokinetic properties of these bispyridinium compounds.
We also report our preliminary investigations into the mode of
action of this class of antifungal compound.
Results and Discussion
C5H11
N
Chemistry. In previous studies, we found that 1,12-
bis(pentylpyridinium)dodecane 1 had broad spectrum antifungal
activity with low haemolytic activity and low cytotoxicity against
mammalian cells.14 Therefore, a series of compounds were
designed based on the structure of 1, in which the
pentylpyridinium headgroups were held constant while the linker
between the two headgroups was varied. Compounds 1-5, in
which the alkyl chain length was varied from eight to sixteen
methylene units, were readily prepared upon reaction of the
appropriate a,w-dibromoalkane with 2.2 equivalents of 4-
pentylpyridine 6 (Scheme 1). While 2 and 3 were solids and could
be purified by recrystallisation as the dibromide salts, compounds
1, 4 and 5 were purified by column chromatography, followed by
ion exchange to give the dichloride salts. We have previously
established that changing the counterion from bromide to chloride
has no effect on the biological activity of these cationic
compounds.12 To probe the effect of the hydrophobicity of the
spacer between the headgroups, compounds 7 and 8, in which
the alkyl chain was replaced by ethylene glycol, were prepared in
a similar manner from 1,11-dibromo(tetraethyleneglycol)16 and
1,14-dibromo(pentaethyleneglycol),17 respectively.
N
C5H11
9
2 Cl
2 Cl
C5H11
N
N
10
C5H11
C5H11
N
N
C5H11
2 Cl
11
C5H11
N
N
C5H11
C5H11
12
2 Cl
2 Cl
N
N
13
C5H11
Figure 1. Structures of compounds 9-13.
Br
Br
n
C5H11
The linkers required for synthesis of 9-11 containing either one
alkene of defined stereochemistry [(E)- or (Z)-] or an alkyne
between the central carbon atoms of the spacer unit were
prepared from a common precursor. Hept-3-yn-1-ol was treated
with sodium hydride and diaminopropane in an alkyne ‘zipper’
reaction18 to give the terminal alkyne 30 (Scheme 2). Following
protection of the alcohol with a tetrahydropyranyl (THP) ether, the
alkyne 3119 was treated with n-butyllithium and the resulting
alkynoate was reacted with the known bromide 3220 to provide the
symmetrically substituted alkyne 33. Removal of the THP groups
upon treatment with p-toluenesulfonic acid gave the diol 34.
Stereoselective reduction of the alkyne to provide the (E)-alkene
35 was achieved upon treatment of 34 with lithium metal in
ammonia.21 Unfortunately, hydrogenation of 34 in the presence of
the Lindlar catalyst was not stereoselective, giving a 1.6:1 mixture
of the (Z)- and (E)-alkenes (36). This mixture was used in the
subsequent formation of the bis(pentylpyridinium) salt.
82 - 86%
N
6
C5H11
N
N
2X
n
C5H11
1: n = 3, X = Br
2: n = 1, X = Br
3: n = 2, X = Cl
4: n = 4, X = Cl
5: n = 5, X = Cl
O
O
Br
O
Br
m
C5H11
75 - 90%
N
To convert alcohols 34-36 to the bis(pentylpyridinium)
compounds 9-11, the alcohols were treated with p-
toluenesulfonylchloride to provide the intermediate ditosylates 37-
39, which were then reacted with 4-pentylpyridine in a similar
manner to that described for the dibromides above. Ion exchange
provided 9-11 as their dichloride salts. Compounds 12 and 13,
which incorporate diyne linkers and therefore have increased
6
2X
O
O
N
O
N
m
C5H11
C5H11
7: m = 1, X = Cl
8: m = 2, X = Cl
Scheme 1. Synthesis of the bis(pentylpyridinium) compounds 1 - 5 and 7 - 8.
2
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10.1002/cmdc.201800331
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FULL PAPER
i)
ii)
Br
OTHP
HO
THPO
+
HO
83 %
70 %
31
30
32
iii)
THPO
RO
iv)
60 %
vi)
OR
RO
90 %
35: R = H
38: R = Ts
THPO
(viii)
RO
33
34: R = H
v)
(viii)
37: R = Ts
98 %
RO
RO
Z:E (1.6:1)
36: R = H
39: R = Ts
(viii)
Scheme 2. Synthesis of linkers 37-39.
F
F
F
F
C5H11
N
N
C5H11
C5H11
N
N
N
C5H11
2 Cl
C5H11
14
2 Cl
C5H11
22
N
F
2 Cl
F
N
15
16
2 Br
C5H11
N
F
F
23
C5H11
2 Cl
N
N
N
N
2 Cl
C5H11
C5H11
S
N
N
N
N
24
2 Cl
C5H11
C5H11
17
C5H11
C5H11
S
25
C5H11
C5H11
2 Cl
C5H11
18
2 Cl
N
N
N
C5H11
2 Cl
26
C5H11
N
C5H11
C5H11
N
19
C5H11
2 Cl
N
N
27
N
2 Cl
C5H11
C5H11
2 Cl
N
C5H11
28
C5H11
N
N
20
N
2 Cl
C5H11
C5H11
C5H11
N
C5H11
N
N
29
N
C5H11
C5H11
2 Cl
21
2 Cl
Figure 2. Structures of compounds 14-29
3
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10.1002/cmdc.201800331
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rigidity and a more linear shape than 9-11 were prepared in a
similar manner from the known precursors 1,10-dihydroxy-dec-
4,6-diyne,22 and 1,12-dihydroxy-dodec-5,7-diyne,23 respectively.
The synthesis of the required linkers for compounds 14-29 (Figure
2) was achieved from appropriately substituted diiodo-aromatic
precursors. A range of aromatic cores 40-46 was used to provide
linkers with different geometries and substitution patterns.
Treatment of 40-46 with either 5-hexyn-1-ol or 4-pentyn-1-ol in
double Sonogashira couplings24,25 allowed the preparation of
linkers 47-54 in which the rigid aromatic region of the linker was
extended by conjugation to alkynes (Scheme 3). Subsequent
hydrogenation of the triple bonds provided efficient access to
more flexible analogues 55-62. In all cases, the resulting diols
were converted into either the dibromides or ditosylates and
further reacted with 2.2 equivalents of 4-pentylpyridine as
described above to yield the bis(pentylpyridinium) salts 14-29.
against both yeasts, C. albicans and C. neoformans, was
observed for many of these compounds. For the series 1-5, in
which the length between the headgroups was increased (from
eight to sixteen methylene units), while the spacer was
maintained as an alkyl chain, antifungal activity increased against
both C. albicans and C. neoformans with increased spacer length.
This trend was clearest for C. neoformans with the MIC
decreasing significantly (44 µM for 2 to 0.7 µM for 5). However,
increasing the hydrophilicity of the spacer by replacing the alkyl
chains with polyethylene glycol derivatives (compounds 7 and 8)
resulted in a loss of antifungal activity. Rigidifying the spacer
between the headgroups through the incorporation of bis-alkynes,
while maintaining the same overall chain length of 10-12 carbon
atoms (12 vs 3 (10 carbon spacer) and 13 vs 1 (12 carbon spacer),
respectively) also resulted in a significant loss of activity although
the same trend for higher activity for the compound containing the
12 carbon spacer over that with the 10 carbon spacer was
observed. Comparison of the antifungal activity for compounds 1,
9, 10 and 11, in which the spacer length was maintained at 12
carbons, but the bond between the central two carbon atoms was
varied between alkane, alkene (both E and Z geometries) and
alkyne, suggests that a small amount of rigidity at this position is
tolerated with activity of both alkyne-containing 9 and Z-alkene
containing 11 being within two dilution steps of that of 1. However,
10 with the E-alkene spacer has the lowest antifungal activity of
this group of compounds indicating that, despite the flexibility of
these chains, the geometry of the alkene is important and
suggesting that placing a kink in the spacer (as for Z-alkene
containing 11) might lead to increased antifungal activity. We
therefore synthesised the series of compounds 14-29 to further
explore these tentative structure-activity relationships.
We first prepared a series of compounds (20, 21, 14 and 15) with
spacer lengths approximately equivalent to those of the alkyl
chain spacers of 4 and 5 (fourteen and sixteen methylene units,
respectively), but with differing rigidity, through the incorporation
of a para-substituted benzene ring in the centre of the spacer,
flanked by alkynes in the case of 20 and 14. In comparison to 4,
both 20 and 21 exhibit slightly decreased antifungal activity
against both C. albicans and C. neoformans, while the slightly
longer chain compounds 14 and 15 have similar activity to 5
against C. neoformans, but slightly reduced activity against C.
albicans. In this compound series, the additional rigidity imparted
by the flanking alkynes did not substantially alter the observed
antifungal activity. As was observed for the alkyl chain series,
increasing the length of the spacer (14 and 15 vs 20 and 21)
resulted in an increase in antifungal activity.
I
I
HO
Ar
+
n
i)
HO
Ar
n
n
47-54
OH
HO
ii)
n
Ar
55-62
n = 1, 2
n
OH
I
Ar
I
I
I
I
I
I
I
I
40
41
42
F
F
F
I
I
I
I
45
46
F
43
I
I
Altering the substitution pattern of the central benzene ring of the
spacer from para- (14, 15) to meta- (16, 17) and ortho- (18, 19)
while maintaining the overall spacer length and rigidity did not
have a significant effect on activity against C. neoformans and C.
albicans. However, a notable increase in activity against A.
fumigatus was observed for the ortho-substituted compounds,
with 19 the only compound tested to show strong activity against
this mold. Replacing the para-substituted benzene spacer of 14
and 15 with either a para-substituted tetrafluorobenzene (22, 23)
or 2,5-thiophene spacer (24, 25) did not result in significant
changes in antifungal activity, suggesting that the polarity of this
aromatic group is not important for activity.
I
S
44
Scheme 3. Synthesis of linkers 47-62.
Antifungal Activity. Compounds 1-5 and 7-29 were assayed by
a standardised serial dilution microtitre sensitivity test for their
antifungal activities against reference strains of Cryptococcus
neoformans, Candida albicans and Aspergillus fumigatus (Table
1).26,27 All compounds were fully soluble in the assay media at
twice the highest concentrations tested. While none of the
compounds, with the exception of 19, exhibited significant
antifungal activity against the mold, A. fumigatus, strong activity
Compounds 26-29 contain larger and more rigid aromatic groups
(biphenyl and fluorene) in the centre of the spacers with shorter
alkyl substituents, such that the overall length of these spacers is
4
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10.1002/cmdc.201800331
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FULL PAPER
comparable to that of 5, 14 and 15. These compounds have
slightly reduced antifungal activity, particularly in the case of 26
and 28 in which the flanking alkynes provide an extended rigid
core, suggesting that while linear rigidity in the spacers is
tolerated to some extent, some flexibility is required.
was replaced by tetrafluorophenyl and thiophene cores,
respectively shows that the modified aromatics provide a slight
improvement in haemolytic activity. Overall, the incorporation of
aromatic rings into the spacers results in slightly increased
haemolytic activity over that observed for compounds with alkyl
spacers of comparable chain length, with the exception of the
ortho-substituted derivatives.
Table 1. Influence of linker structure on in vitro antifungal potency, reported as
MIC (µM).
Table 2 Influence of linker structure on haemolytic activity and mammalian
cytotoxicity.
C. albicans
(ATCC 10231)
A. fumigatus
(ATCC 204305)
C. neoformans
(ATCC 90112)
Compound
X-
CC50 (µM)[c]
A549
CC50 (µM)[c]
MDCK
HC50 (µM)[b]
2
3
1[a]
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Cl
Cl
Cl
Cl
Cl
44
11
2.7
2.7
nd
nd
Compound
X-
Erythrocytes
2
Br
Br
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
>350
>350
>350
>350
250
nd
nd
nd
nd
1.4
1.4
0.7
88
2.7
88
3
1[a]
4
0.7
nd
110±7
52±12
9.6±0.9
>350
nd
153±8
41±10
15±5
>350
nd
5
0.7
nd
4
7
>175
>175
5.5-11
44
nd
5
8
88
nd
9
>350
>350
nd
9
2.7
22
175
>350
>350
>100
>100
100
50
10
12
13
15
16
17
18
19
20
21
22
24
25
27
29
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
>350
>350
45±7
nd
>350
>350
68±6
nd
5.5
100
25
11
nd
100
50
109
95
1.5
0.8
1.5
0.8
1.3
1.3
6.2
6.2
1.3
1.3
0.7-1.3
0.7-1.3
22
6.2
175
36±8
70±8
>350
116±13
74±12
43±12
80±10
62±6
nd
60
6.2
>350
>350
>350
>350
182
120±9
>350
140±10
165
6.2
50
6.2
50
5.5-11
2.7
44
11
60±8
105±8
113±9
nd
12.5
12.5
1.3
100
50
233
194
44
90
1.3
88
>350
nd
nd
1.3-2.7
1.3
88
88
[a] The activity of this compound has been reported previously.14 [b] Assays
were performed in triplicate and in all cases standard errors were <5%. [c] Data
are presented as the meansꢀ±ꢀSD of 3 independent experiments.
22
>350
>350
>350
11
11
22
22
Cl
5.5
2.7
88
29
Cytotoxicity. We subsequently evaluated the broader
cytotoxicity of this subset of compounds against two mammalian
cell lines [human lung epithelial cells (A549) and Madin-Darby
Canine Kidney cells (MDCK)] using an MTS assay (Table 2).28 In
general, mammalian cell cytotoxicity of the compounds correlated
with their haemolytic activity, suggesting that the cytotoxicity may
be a result of cell lysis. Increasing the length of the spacer
between the two pyridinium head groups resulted in a significant
increase in cytotoxicity (CC50 against A548 cells = 115 µM for 1
compared with 10 µM for 5) and this trend was maintained when
a para-substituted phenyl group was incorporated in the spacer.
Incorporation of a para- or meta- substituted phenyl spacer (15
and 17, respectively) or a 2,5-substituted thiophene (25) within
the linker led to a slight decrease in cytotoxicity in comparison to
a compound containing an alkyl chain of approximately the same
length (5), whereas the inclusion of an ortho-substituted phenyl
ring resulted in a significant decrease in cytotoxicity (19; CC50
>350 mM for both A549 and MDCK cell lines). The majority of
strongly antifungal compounds exhibited cytotoxicity at levels well
above their MIC values against fungi, with therapeutic indices
(calculated as CC50 (MDCK) / MIC50 against C. neoformans) in the
range 14 - >107.
[a] The activity of this compound has been reported previously.14
Haemolytic assays. Since several of the compounds exhibited
strong antifungal activity against both C. albicans and C.
neoformans, we next evaluated a selected subset of compounds
for their haemolytic activity (Table 2), since this compound class
bears some structural resemblance to bolaform surfactants.
Compounds 2- 4, 9, 10, 18-21 and 29 did not lyse erythrocytes at
any of the concentrations tested (up to 350 µM). However, some
haemolysis was observed for compounds where the alkyl chain
length between the head groups was extended to 16 carbons
(compound 5) and for compounds 15-17, 22, 24, 25 and 27
bearing aromatic groups in the linker. Haemolytic activity varied
with the substitution pattern on the benzene spacer with the para-
and meta-substituted derivatives 15 and 17 exhibiting higher
haemolytic activity than the ortho- (19) substituted compound and
a similar trend observed for the meta- and ortho-substituted
derivatives, 16 and 18, with extended alkyne cores. Shortening
the length of the spacer resulted in lower haemolytic activity (HC50
= 109 µM for 15, vs >350 µM for 21). A comparison of compounds
14, 22 and 24 in which the para-substituted benzene core of 14
5
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Phospholipase inhibition. Given that bis-cationic compounds,
These strains had either normal (H99 and Dplb1::REC15),
such as 1, had previously been found to inhibit cryptococcal PLB1, significantly reduced (Dsec14-1) or absent (Dplb1) PLB1 secretion.
we evaluated the inhibitory activity of selected compounds
against both cryptoccocal PLB1 and a representative mammalian
phospholipase, porcine pancreatic PLA2 (ppPLA2) (Table 3) to
evaluate whether this might be their mode of antifungal action.
Crude secretions, in which the phospholipase activity is
attributable to PLB1,9 were collected from concentrated
cryptococcal cell suspensions following an overnight incubation.
Inhibition assays against its three activities (PLB: Phospholipase
B; LPL: lysophospholipase and LPTA: lysophospholipase
acyltransferase) were then carried out at pH 4, under previously
optimised conditions.29 Inhibition of ppPLA2 was determined using
the method of De Haas et. al.30 Compounds were initially tested
at concentrations of 250, 25, 2.5 and 0.25 µM against both
ppPLA2 and cryptococcal PLB1, and compounds which showed
inhibition were re-assayed over an appropriate concentration
range to determine their IC50s.
None of the compounds exhibited significant inhibition of the LPL
or LPTA activities of PLB1 at any of the concentrations tested,
however some compounds showed significant inhibition of the
PLB activity of this enzyme. Increasing the length of the alkyl
spacer from 12 to 14 methylene units resulted in an approximately
2.5–fold increase in inhibition of PLB activity. In general, the
incorporation of an aromatic group into the spacer resulted in
increased inhibition of PLB, in comparison to that exhibited by 1.
However, in contrast to the low inhibition of ppPLA2 activity
observed for 1 and related compounds with a 12 carbon spacer,14
significant inhibition of ppPLA2 was also observed for the majority
of compounds containing an aromatic group in the spacer. The
exception to this was compound 24, containing a thiophene group
No significant differences were observed in the susceptibility of
these mutants to 1 (MIC50 = 1.4 µM in all cases, Figure S1),
confirming that the antifungal activity of these compounds is
unlikely to be a result of inhibition of PLB1.
Disruption of mitochondrial function. The bispyridinum
compounds 1-5 and 7-29 are lipophilic cations, similar in structure
to compounds known to disrupt mitochondrial function by altering
mitochondrial membrane potential, such as the mitochondrial
poison, dequalinium.32 It has recently been suggested that
disruption of mitochondrial function in fungi might provide a novel
target for antifungal drug discovery,33 and there are several
reports of antifungal activity that is correlated with disruption of
mitochondrial function.34 In efforts to determine the mode of
antifungal action of the bispyridinium compounds, we performed
preliminary investigations of their effect on mitochondrial
membrane potential. We selected two compounds for this
purpose; 1 which shows strong antifungal activity against both C.
albicans and C. neoformans and 8, which has reduced lipophilicity
and did not exhibit antifungal activity.
in the spacer, which exhibited significant inhibition of PLB (IC50
=
6 µM), but did not inhibit ppPLA2 at any of the concentrations
tested (IC50 >250 µM).
Table 3. Inhibition of fungal PLB and ppPLA2 by bis(alkylpyridinium)alkanes.
Compound
ppPLA2 IC50
PLB IC50
(µM)
(µM)
1[a]
4
104
39
13
27
7
>250
130
25
Figure 3. Assessment of mitochondrial membrane potential by flow cytometry
using DiOC6. Fungal cells were incubated with 2-4 µg/ml 1 or 2-4 µg/ml 8 or 3
µM FCCP for 3.5 hours at 30oC and stained with DiOC6 (0.3 µM). After 20 min
of staining at ambient temperature, the incorporation of the dye was quantified
by flow cytometry. (A) Average DiOC6 fluorescence. Error bars indicate
standard deviation. The assay was performed in triplicate. (B) Histograms
illustrating accumulation of DiOC6 across fungal cell populations following
treatments as indicated.
9
14
15
18
19
22
24
29
24
35
18
12
17
6
128
27
25
>250
16
6
The effect on these two compounds on mitochondrial function was
evaluated in C. neoformans by staining with 3,3'-
[a] The activity of this compound has been reported previously.14
dihexyloxacarbocyanine iodide (DiOC6),
a
cell-permeable,
fluorescent, lipophilic dye which accumulates in highly polarized
membranes. At low concentration, DiOC6 accumulation was
shown to reflect mitochondrial membrane potential.35 Fungal cells
were treated with the compounds 1 and 8, and a potent oxidative
While most of the compounds evaluated had increased inhibitory
activity against fungal PLB1 compared to 1, there was no strong
correlation between their antifungal activity and PLB1 inhibitory
activity, suggesting that this may not be the primary mode of
antifungal action. This was further supported by evaluation of the
antifungal activity of 1 against four strains of C. neoformans with
differing levels of PLB1 secretion, strain H99 and the three mutant
strains created from it: Dplb1, Dplb1::REC15 and Dsec14-1.9,31
phosphorylation
uncoupler,
carbonyl
cyanide-4-
(trifluoromethoxy)phenylhydrazone (FCCP) as
a
control for
compromised mitochondrial function.36 DiOC6 accumulation was
assessed using flow cytometry. Figure 3 shows that untreated
6
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10.1002/cmdc.201800331
ChemMedChem
FULL PAPER
cells and cells exposed to 8 accumulate similar amount of DiOC6.
However, treatment with compound 1 and FCCP caused
reduction in DiOC6 accumulation. Furthermore, compound 1
affects DiOC6 accumulation in a dose-dependent manner. These
findings are consistent with a highly specific, detrimental effect of
the compound 1 on fungal mitochondrial potential, similar to that
of FCCP.
towards mammalian cells, leading to decreased therapeutic
indices. Replacement of the alkyl spacer by more hydrophilic
ethylene glycol derivatives resulted in a complete loss of
antifungal activity. The incorporation of aromatic groups into the
alkyl spacer resulted in changes to antifungal activity, haemolytic
activity and mammalian cytotoxicity, with larger, more rigid groups
resulting in
a reduction in antifungal activity, while the
To further assess the impact of 1 and 8 on fungal mitochondrial
function, the morphology of treated cells and their DiOC6 staining
pattern were assessed by microscopy. Cell nuclei were visualized
by staining with DAPI. In control cells and cells treated with 8 (4
µg/ml) for 7 hours, brightly stained punctate mitochondrial
structures were observed by DiOC6 staining, indicative of
functional mitochondria with strong membrane potential (Figure
4).37 In contrast, cells treated with 1 (4 µg/ml) for 7 hours were
significantly enlarged and the punctate mitochondrial structures
were no longer visible, indicative of mitochondrial damage and
disruption of mitochondrial membrane potential. Consistent with
the flow cytometry data, the intensity of DiOC6 staining in cells
treated with 1 was dramatically reduced.
incorporation of an ortho-substituted benzene group into the
spacer gave improved activity against A. fumigatus, whilst activity
against C. neoformans and C. albicans was maintained. Both
haemolytic activity and mammalian cytotoxicity were reduced in
comparison to compounds with alkyl spacers of equivalent length,
providing compounds with increased therapeutic indices.
While some of the compounds inhibited the PLB activity of fungal
PLB1, there was a poor correlation between antifungal potency
and enzyme inhibition, indicating that inhibition of PLB1 is unlikely
to be the major mode of antifungal action of these compounds.
This was supported by the lack of variation in MIC50 when the
antifungal activity of compound 1 was evaluated against four
strains of C. neoformans with either normal (H99 and
Dplb1::REC15), significantly reduced (Dsec14-1) or absent
(Dplb1) PLB1 secretion levels. However, preliminary studies
suggest that these compounds may act via disruption of
mitochondrial function, since treatment of C. neoformans with
compound 1, which exhibits strong antifungal activity, resulted in
mitochondrial damage and loss of mitochondrial membrane
potential, as evidenced by staining cells treated with 1 with DiOC6.
In contrast, compound 8, which does not display antifungal activity,
did not appear to affect mitochondrial function in C. neoformans.
This opens up a new avenue for investigation of the antifungal
activity of these and related biscationic compounds.
Experimental Section
Chemistry. Melting points were determined using a Gallenkamp melting
point apparatus and are uncorrected. NMR spectra were recorded on a
Bruker Avance DPX 200 or a Bruker Avance DPX 300 spectrometer. The
solvent 1H and 13C signals, dH 7.26 for residual CHCl3 and dC 77.0 for
CDCl3, dH 3.31 and dC 49.0 for CD3OD, dH 2.50 and dC 39.5 for d6-DMSO
were used as internal references. Low resolution mass spectra were
recorded on a Finnigan LCQ ion trap mass spectrometer (ESI and APCI).
High resolution mass spectra were recorded on a Bruker 7T Fourier
Transform Ion Cyclotron Resonance Mass Spectrometer (ESI and APCI).
Analytical thin layer chromatography (TLC) was performed using
precoated silica gel plates (Merck Kieselgel 60 F254) and visualized using
a basic KMnO4 staining reagent. Preparative column chromatography was
carried out using Merck Kieselgel 60 silica gel (SiO2, 0.04 – 0.065 mm) or
neutral alumina (activity II – III) with the indicated solvent systems (v/v).
Ion exchange chromatography was performed on Lewatit MP-64 resin
(chloride form), which was swollen in H2O, washed with 3 bed volumes of
0.1 M HCl (aq) and then washed with H2O until the washings were neutral.
Analytical RP-HPLC was performed using an XTerraRP C18 column (5
µm, 4.6 mm ID, 250 mm) with a flow rate of 0.2 mL/min. Preparative RP-
HPLC was performed using an XTerraRP C18 column (10 µm, 22 mm ID,
Figure 4. Fluorescence microscope images of C. neoformans cells stained with
DiOC6 (to visualise mitochondrial membrane potential) and DAPI (to visualise
fungal nuclei). Cells were incubated with no drug (untreated), compound 1 (4
µg/mL) or compound 8 (4 µg/mL) for 7 h at 30 oC prior to imaging. Due to the
weak staining of 1-treated cells with DiOC6, image acquisition time and
brightness of the resulting images were adjusted to allow comparison of the
DiOC6 staining pattern with the control cells. DIC - differential interference
contrast.
Conclusions
The biscationic 1,12-bis(4'-pentylpyridinium)dodecane has
previously been reported to exhibit antifungal activity against a
range of yeasts (but not molds).14,15 Using this compound as a
starting point, a series of bis(4-pentylpyridinium)s, in which the
spacer between the two headgroups was varied, were designed
and synthesised to probe the antifungal structure-activity
relationship of this class of compounds. The haemolytic activity
300
mm)
with
a
flow
rate
of
7.0
mL/min.]
1,11-
Dibromotetraethyleneglycol,16 1,14-dibromopenta(ethylene glycol),17 1,10-
dihydroxy-dec-4,6-diyne,22 1,12-dihydroxy-dodec-5,7-diyne,23 1,4-bis(6'-
hydroxy-1'-hexynyl)benzene,25 1,4-bis(5’-hydroxy-1'-pentynyl)benzene,25
1,4-bis(5'-hydroxypentyl)benzene,25 1,4-bis(6'-hydroxyhexyl)benzene,25
1,4-bis(6'-bromohexyl)benzene,38 compounds 1,14 6,39 31,19 and 32,20 were
prepared according to literature methods. Detailed synthetic methods for
precursors are provided in the supporting information.
and mammalian cell cytotoxicity of
a selected group of
compounds were also evaluated, as was their ability to inhibit
fungal PLB and mammalian PLA2. It was found that lengthening
the alkyl spacer between the two headgroups from twelve to
sixteen methlyene units resulted in increased antifungal potency
against C. neoformans and C. albicans, but this was accompanied
by significant increases in haemolytic activity and cytotoxicity
7
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10.1002/cmdc.201800331
ChemMedChem
FULL PAPER
pentanone (2.0 mL) and 6 (0.52 g, 3.50 mmol) was added. The mixture
was stirred at reflux for 18 h under a nitrogen atmosphere, then the solvent
was removed under reduced pressure. The residue was then diluted with
H2O (~ 15 mL) and washed with Et2O (3 x 20 mL). The aqueous layer was
extracted with CH2Cl2 (3 x 20 mL), then the combined CH2Cl2 phases were
concentrated under reduced pressure. The residue was purified by
chromatography on neutral Al2O3 (activity II-III), (98:2 CHCl3/MeOH →
90:10 CHCl3/MeOH). The required fractions were combined and the
solvent removed under reduced pressure. The residue was passed down
a column of Lewatit MP-64 anion resin (Cl-), eluting with EtOH. The
resulting fractions were combined and the solvent removed under reduced
pressure to give 4 as a pale brown waxy oil (0.67 g, 85 %). 1H NMR (300
MHz, CD3OD): δ 8.94 (4H, d, J = 6.5 Hz), 8.02 (4H, d, J = 6.5 Hz), 4.65
(4H, t, J = 7.5 Hz), 3.02 (4H, t, J = 7.5 Hz), 2.07 (4H, m), 1.82 (4H, m), 1.42
(28H, m), 0.98 (6H, m); 13C NMR (75 MHz, CD3OD): δ 164.2, 144.1, 128.5,
61.2, 35.6, 31.6, 31.5, 29.8, 29.7, 29.6, 29.2, 26.2, 22.5, 13.6, 1 signal
obscured or overlapping; m/z ESI (positive ion) 247 [M-2Cl-]2+ (100 %), 494
[M-2Cl--H+]+ (80), 529 [M-37Cl-]+ (60), 531 [M-35Cl-]+ (25). Found [M-2Cl-]2+
247.2293, [C34H58N2]2+ requires 247.2294. Anal. (C34H58Cl2N2·2.5H2O)
calcd, C 66.8; H 10.3; N 4.6. Found C 66.8; H 9.8; N 4.6.
General methods
General method for bromination Carbon tetrabromide (1.1 eq per
hydroxyl group) was added to a solution of alcohol (1 eq.) in dry CH2Cl2 (1
mL / 15 mg substrate) and the resulting mixture was stirred at RT for 10
min. Triphenylphosphine (1.1 eq per hydroxyl) in dry CH2Cl2 (1 mL / 50 mg
triphenylphosphine) was added to the solution at 0 °C and the mixture was
allowed to warm to RT and stirred until complete by TLC (5-9 h) under a
nitrogen atmosphere. The solvent was removed under reduced pressure
then hexane or hexane/EtOAc (1:1 v/v) was added to the residue. The
resulting solution was washed with NaHCO3 (2 x 30 mL) and brine (2 x 30
mL), dried (MgSO4) and the solvent was removed under reduced pressure.
The residue was triturated with hexane then filtrates were collected, and
the solvent was removed under reduced pressure. The filtrate was
collected and the solvent was removed under reduced pressure. The
residue was purified by flash chromatography to give the desired
dibromide product.
General method for the preparation of bispyridinium salts The
dibromide or ditosylate (1 eq) was dissolved in 4-methyl-2-pentanone
(1 mL per 50 mg dibromide) and 6 (2.2 eq. per 1 eq. dibromide) was added.
The mixture was stirred at reflux for 15 - 18 h under a nitrogen atmosphere
then the solvent was removed under reduced pressure and the residue
was triturated with Et2O or EtOAc (8 ´ 10 mL), then purified by
chromatography on neutral Al2O3 (activity II-III), (98:2 CHCl3/MeOH →
90:10 CHCl3/MeOH). The required fractions were combined and the
solvent removed under reduced pressure. The residue was passed down
a column of Lewatit MP-64 anion resin (Cl-), eluting with EtOH. The
required fractions were combined and the solvent removed under reduced
pressure.
1,16-Bis(4'-pentylpyridinium)hexadecane
dichloride
(5)
1,16-
Dibromohexadecane (0.20 g, 0.49 mmol) was dissolved in 4-methyl-2-
pentanone (2.0 mL) and 6 (0.18 g, 1.24 mmol) was added. The mixture
was stirred at reflux for 18 h under a nitrogen atmosphere, and the solvent
was removed under reduced pressure. The residue was then diluted with
H2O (~ 15 mL) and washed with Et2O (3 x 20 mL). The aqueous layer was
extracted with CH2Cl2 (3 x 20 mL), then the combined CH2Cl2 phases were
concentrated under reduced pressure. The residue was purified by
chromatography on neutral Al2O3 (activity II-III), (98:2 CHCl3/MeOH →
90:10 CHCl3/MeOH). The required fractions were combined and the
solvent removed under reduced pressure. The residue was passed down
a column of Lewatit MP-64 anion resin (Cl-), eluting with EtOH. The
resulting fractions were combined and the solvent removed under reduced
pressure to give 5 as a pale brown waxy oil (0.25 g, 86 %). 1H NMR (300
MHz, CD3OD): δ 8.99 (4H, d, J = 6.5 Hz), 8.02 (4H, d, J = 6.5 Hz), 4.66
(4H, t, J = 7.5 Hz), 2.98 (4H, t, J = 7.5 Hz), 2.03 (4H, m), 1.79 (4H, m), 1.41
(32H, m), 0.93 (6H, m); 13C NMR (75 MHz, CD3OD): δ 164.0, 144.3, 128.5,
61.2, 35.6, 31.6, 31.5, 29.9, 29.8, 29.6, 29.5, 29.2, 26.3, 22.5, 13.5, 1
signal obscured or overlapping; m/z ESI (positive ion) 261 [M-2Cl-]2+
(100 %), 521 [M-2Cl--H+]+ (90), 557 [M-37Cl-]+ (45), 559 [M-35Cl-]+ (20).
Found [M-2Cl-]2+ 261.2454, [C36H62N2]2+ requires 261.2451. Anal.
(C36H62Cl2N2·2.5H2O) calcd, C 67.6; H 10.6; N 4.4. Found C 67.3; H 10.4;
N 4.3.
1,8-Bis(4'-pentylpyridinium)octane dibromide (2) 1,8-Dibromooctane
(0.50 g, 1.84 mmol) was dissolved in 4-methyl-2-pentanone (2.0 mL) and
6 (0.68 g, 4.56 mmol) was added. The mixture was stirred at reflux for 18
h under a nitrogen atmosphere, and the solvent was removed under
reduced pressure. The crude product was dissolved in methanol (25 mL)
and decolourising charcoal was added, then the mixture was boiled for 5
min. The charcoal was removed by filtration and the solvent removed
under reduced pressure. The residue was recrystallised from MeOH/Et2O
to yield 2 as a colourless solid (0.87 g, 83 %); m.p. 190 – 191 ºC; 1H NMR
(300 MHz, d6-DMSO): δ 9.00 (4H, d, J = 6.5 Hz), 8.03 (4H, d, J = 6.5 Hz),
4.54 (4H, t, J = 7.6 Hz), 2.87 (4H, t, J = 7.5 Hz,), 1.89 (4H, m), 1.66 (4H,
m), 1.29 (16H, m), 0.86 (6H, m); 13C NMR (75 MHz, d6-DMSO): δ 163.4,
144.9, 128.5, 60.7, 35.4, 31.5, 31.4, 29.6, 29.0, 26.2, 22.6, 14.6; m/z ESI
(positive ion) 205 [M-2Br-]2+ (100 %), 489 [M-81Br-]+ (15), 491 [M-79Br-]+
(15). Found [M-81Br-]+ 489.2850, [C28H46N2Br]+ requires 489.2839. Anal.
(C28H46Br2N2) calcd, C 58.95; H 8.1; N 4.9. Found C 58.9; H 8.0; N 4.9.
1,11-Bis(4'-pentylpyridinium)tetra(ethyleneglycol)
dichloride
(7)
Treatment of the 1,11-dibromotetraethyleneglycol (0.21 g, 0.65 mmol) and
6 (0.20 g, 1.38 mmol) according to the general method for bispyridinium
formation gave 7 as a pale yellow waxy oil (0.31 g, 90 %). 1H NMR (300
MHz, CD3OD): δ 8.96 (4H, d, J = 6.5 Hz), 8.04 (4H, d, J = 6.5 Hz), 4.87
(4H, t, J = 7.0 Hz), 4.04 (4H, t, J = 7.4 Hz), 3.63 (4H, m), 3.54 (4H, m), 2.99
1,10-Bis(4'-pentylpyridinium)decane
dibromide
(3)
1,10-
Dibromodecane (0.50 g, 1.66 mmol) was dissolved in 4-methyl-2-
pentanone (2.0 mL) and 6 (0.69 g, 4.20 mmol) was added. The mixture
was stirred at reflux for 18 h under a nitrogen atmosphere, and the solvent
was removed under reduced pressure. The crude product was dissolved
in methanol (25 mL) and decolourising charcoal was added, then the
mixture was boiled for 5 min. The charcoal was removed by filtration and
the solvent removed under reduced pressure. The residue was
recrystallised from MeOH/Et2O to yield 3 as a colourless solid (0.82 g,
82 %); m.p. 173 – 174 ºC; 1H NMR (300 MHz, d6-DMSO): δ 9.01 (4H, d, J
= 6.5 Hz), 8.03 (4H, d, J = 6.5 Hz), 4.55 (4H, t, J = 7.5 Hz), 2.87 (4H, t, J =
7.5 Hz), 1.89 (4H, m), 1.65 (4H, m), 1.29 (20H, m), 0.86 (6H, m); 13C NMR
(75 MHz, d6-DMSO): δ 163.4, 144.8, 128.5, 60.7, 35.4, 31.5, 31.4, 29.6,
29.5, 29.2, 26.3, 22.6, 14.6; m/z ESI (positive ion) 219 [M-2Br-]2+ (100 %),
437 [M-2Br--H+]+ (40), 517 [M-81Br-]+ (40), 519 [M-79Br-]+ (40). Found [M-
81Br-]+ 517.3159, [C30H50N2Br]+ requires 517.3152. Anal. (C30H50Br2N2)
calcd, C 60.2; H 8.4; N 4.7. Found C 60.3; H 8.5; N 4.6.
(4H, t, J = 6.4 Hz), 1.70 (4H, m), 1.40 (8H, m), 0.96 (6H, t, J = 6.6 Hz); 13
C
NMR (75 MHz, CD3OD): δ 164.4, 144.9, 127.9, 70.5, 69.4, 65.9, 60.8, 35.9,
31.5, 29.8, 22.5, 14.8; m/z ESI (positive ion) 229 [M-2Cl-]2+ (100 %), 457
[M-2Cl--H+]+ (55), 493 [M-35Cl-]+ (25). Found [M-2Cl-]2+ 229.1750,
[C28H46N2O3]2+ requires 229.1749. Anal. (C28H46Cl2N2O3·2H2O) calcd, C
59.5; H 8.9; N 4.95. Found C 59.45; H 9.0; N 5.0.
1,14-Bis(4'-pentylpyridinium)penta(ethyleneglycol) dichloride (8)
Treatment of the 1,14-dibromopenta(ethyleneglycol) (0.23 g, 0.65 mmol)
and
6 (0.19 g, 1.32 mmol) according to the general method for
bispyridinium formation gave 8 as a pale yellow waxy oil (0.27 g, 75 %).
1H NMR (300 MHz, CD3OD): δ 8.82 (4H, d, J = 6.5 Hz), 7.94 (4H, d, J =
6.5 Hz), 4.75 (4H, m), 3.95 (4H, m), 3.59- 3.49 (12H, m), 2.93 (4H, m),
1.73 (4H, m), 1.35 (8H, m), 0.89 (6H, t, J = 7.7 Hz); 13C NMR (75 MHz,
CD3OD): δ 167.1, 147.4, 130.5, 73.2, 73.0, 72.0, 63.5, 38.3, 34.1, 32.2,
25.0, 15.9, 1 signal obscured or overlapping. MS: m/z ESI (positive ion)
251 [M-2Cl-]2+ (100 %), 502 [M-2Cl--H+]+ (30). Found [M-2Cl-]2+ 251.1879,
1,14-Bis(4'-pentylpyridinium)tetradecane
Dibromotetradecane (0.50 g, 1.40 mmol) was dissolved in 4-methyl-2-
dichloride
(4)
1,14-
8
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10.1002/cmdc.201800331
ChemMedChem
FULL PAPER
[C30H50N2O4]2+ requires 251.1880. Anal. (C30H50Cl2N2O4·2H2O) calcd, C
57.4; H 9.0; N 4.5. Found C 57.4; H 9.0; N 4.4.
Found [M-2Cl-]2+ 231.1981, [C32H54N2]2+ requires 231.1981. Anal.
(C32H50Cl2N2·3H2O) calcd, C 65.4; H 9.6; N 4.8. Found C 65.8; H 9.5; N
4.8.
1,12-Dihydroxydodec-6-yne (34) To a stirred solution of heptynol-THP19
(0.31 g, 1.55 mmol) in THF (2.5 mL) at 0 °C, under an argon atmosphere,
was added n-BuLi (1.40 mL, 2.10 mmol, 2.5 M in hexanes). After 15 min,
HMPA (2.5 mL) was added to the bright yellow solution, then after a further
15 min, a solution of 1-bromo-6-(pentan-5-oxy)tetrahydropyran20 (0.47 g,
1.86 mmol) in THF (1.5 mL) was added. The mixture was allowed to stir at
RT for 8 h then MeOH (0.5 mL) was added to quench excess base, and
the solution was diluted with EtOAc/Hex (20 mL, 1:1) and washed with sat.
NH4Cl (2 x 20 mL) and brine (20 mL). The combined aqueous layers were
extracted with EtOAc/Hex (60 mL, 1:1), the combined organic layers were
dried (MgSO4) and the solvent was removed under reduced pressure. To
the residue were added MeOH (6.0 mL) and p-TsOH (0.10 g, 0.57 mmol),
and the mixture was stirred at 40 °C for 4 h. The solvent was removed
under reduced pressure to give a brown residue that was purified by flash
chromatography (Hex/EtOAc 1:1 → 1:3) and the required fractions were
concentrated to give the diol 34 as a pale yellow liquid (0.18 g, 59 %).
1H NMR (200 MHz, CDCl3): δ 3.64 (4H, t, J= 7.5 Hz), 2.16 (4H, m), 1.62 –
1.50 (12H, m); 13C NMR (50 MHz, CDCl3): δ 80.3, 63.0, 32.4, 28.9, 25.1,
18.8; m/z APCI (positive ion) 199 [M+H]+.
(E)-1,12-Bis(4'-pentylpyridinium)dodec-6-ene dichloride (10)
A
solution of diol 36 (16 mg, 0.08 mmol) in CH2Cl2 (2.5 mL) was cooled to
0 °C then NEt3 (0.04 mL, 0.32 mmol), DMAP (4 mg, cat.) and p-TsCl (46
mg, 0.24 mmol) were added. The resulting solution was stirred under a
nitrogen atmosphere at 4 °C for 4 h, then poured onto a mixture of ice (8
g) and HCl (1 M, 20 mL). The mixture was extracted with CH2Cl2 (3 x 20
mL) and the combined organic phases were washed with sat. aq. NaHCO3
(40 mL) and brine (40 mL), then dried (MgSO4), and the solvent removed
under reduced pressure. The residue was purified by flash
chromatography (Hex/EtOAc 4:0.3 → 4:0.6) and the required fractions
were concentrated to give the tosylate 39 (36 mg, 89 %). 1H NMR
(200 MHz, CDCl3): δ 7.78 (4H, m), 7.35 (4H, m), 5.30 (2H, m), 4.01 (4H, t,
J = 6.5 Hz), 2.44 (6H, s), 1.90 (4H, m), 1.60 (4H, m), 1.29 (8H, m).
Treatment of the ditosylate 39 (36 mg, 0.07 mmol) and 6 (23 mg, 0.15
mmol) according to the general method for bispyridinium formation gave
10 as a brown oil (30 mg, 80 %). 1H NMR (300 MHz, CD3OD): δ 8.85 (4H,
d, J = 6.4 Hz), 7.96 (4H, d, J = 6.4 Hz), 5.41 (2H, m), 4.58 (4H, t, J = 7.5
Hz), 2.96 (4H, t, J = 7.6 Hz), 2.04 (8H, m), 1.75 (4H, m), 1.40 (16H, m),
0.93 (6H, t, J = 6.8 Hz); 13C NMR (75 MHz, CD3OD): δ 164.3, 144.1, 130.4,
128.2, 61.2, 35.5, 32.3, 31.4, 31.2, 29.5, 29.0, 25.6, 22.4, 13.2; m/z ESI
(positive ion) 232 [M-2Cl-]2+ (100 %), 463 [M-2Cl--H+]+ (40), 499 [M-37Cl-]+
(100), 501 [M-35Cl-]+ (37). Found [M-2Cl--H+]+ 463.4050, [C32H52N2]+
requires 463.4047. Anal. (C32H52Cl2N2·4H2O) calcd, C 63.2; H 9.8; N 4.5.
Found C 63.0; H 9.3; N 4.3.
(Z)-1,12-Dihydroxydodec-6-ene (35) To a solution of 34 (37 mg, 0.19
mmol) in MeOH (2.0 mL) was added Lindlar catalyst (Pd-C/CaCO3, 0.13
g). The suspension was stirred under an atmosphere of H2 for 30 min and
filtered through a Celite® pad. Evaporation of the solvent gave a mixture of
the Z alkene 35 and the E alkene 36 (35:36 ratio = 1.6:1, 38 mg, 100 %)
as a colourless oil. Data for Z isomer (35): 1H NMR (300 MHz, CDCl3): δ
5.38 (2H, m), 3.62 (4H, t, J= 7.5 Hz), 2.01 (4H, m), 1.65 – 1.54 (4H, m),
1.37 (8H, m). 13C NMR (75 MHz, CDCl3): δ 130.4, 63.3, 33.0, 29.8, 27.5,
25.9; m/z APCI (positive ion) 201 [M+H]+
(Z)-1,12-Bis(4'-pentylpyridinium)dodec-6-ene dichloride (11)
A
solution of diol 35 (38 mg, 0.19 mmol) in CH2Cl2 (4 mL) was cooled to 0 °C
then NEt3 (0.10 mL, 0.76 mmol), DMAP (4 mg, cat.) and p-TsCl (109 mg,
0.57 mmol) were added. The resulting solution was stirred under a nitrogen
atmosphere at 4 °C for 7 h, then poured onto a mixture of ice (8 g) and HCl
(1 M, 20 mL). The mixture was extracted with CH2Cl2 (3 x 20 mL) and the
combined organic phases were washed with sat. aq. NaHCO3 (40 mL) and
brine (40 mL), then dried (MgSO4), and the solvent removed under
reduced pressure. The residue was purified by flash chromatography
(Hex/EtOAc 4:0.3 → 4:0.6) and the combined fractions were concentrated
to give the tosylate 38 (87 mg, 90 %). Data for Z isomer: 1H NMR (200 MHz,
CDCl3): δ 7.78 (4H, m), 7.33 (4H, m), 5.30 (2H, m), 4.01 (4H, t, J = 6.5 Hz),
2.44 (6H, s), 1.89 (4H, m), 1.63 (4H, m), 1.28 (8H, m). Treatment of the
ditosylate 38 (0.09 g, 0.19 mmol) and 6 (0.06 g, 0.40 mmol) according to
the general method for bispyridinium formation gave 11 as a brown wax
as a 1.6:1 mixture of Z and E isomers as determined by 1H NMR (78 mg,
(E)-1,12-Dihydroxy-dodec-6-ene (36) To a solution of Li (20 mg, 2.65
mmol) in liquid NH3 (12 mL) was added a solution of 34 (35 mg, 0.18 mmol)
in THF (3.0 mL) at -78 °C. The solution was stirred for 12 h at -78 °C, then
the reaction was quenched by the addition of MeOH (1.0 mL). The mixture
was diluted with EtOAc (20 mL), washed with H2O (20 mL) and brine (20
mL), then the organic phase was then dried (MgSO4), and the solvent
removed under reduced pressure. The residue was purified by flash
chromatography (Hex/EtOAc 1:1 → 1:2) and the required fractions were
concentrated to give the diol 36 (32 mg, 90 %) as a colourless oil. 1H NMR
(200 MHz, CDCl3): δ 5.38 (2H, t, J = 7.4 Hz), 3.63 (4H, t, J = 7.5 Hz), 1.99
(4H, m), 1.56 (4H, m), 1.36 (8H, m), (OH not observed). 13C NMR (75 MHz,
CDCl3): δ 130.9, 63.5, 33.2, 33.0, 29.8, 25.7; m/z APCI (positive ion) 201
[M+H]+.
1
77 %). Data for Z isomer: H NMR (300 MHz, CD3OD): δ 8.86 (4H, d, J =
6.4 Hz), 7.96 (4H, d, J = 6.4 Hz), 5.35 (2H, t), 4.59 (4H, t, J = 7.5 Hz), 2.96
(4H, t, J = 7.6 Hz), 2.04 (8H, m), 1.77 (4H, m), 1.40 (16H, m), 0.93 (6H, t,
J = 6.7 Hz); 13C NMR (75 MHz, CD3OD): δ 164.3, 144.1, 130.4, 129.7,
61.2, 35.5, 32.3, 31.3, 29.5, 29.2, 26.9, 25.8, 22.4, 13.2; m/z ESI (positive
ion) 232 [M-2Cl-]2+ (100 %), 463 [M-2Cl--H+]+ (30), 499 [M-37Cl-]+ (80), 501
[M-35Cl-]+ (70). Found [M-2Cl--H+]+ 463.4050, [C32H52N2]+ requires
463.4047. Anal. (C32H52Cl2N2·2.5H2O) calcd, C 66.1; H 9.7; N 4.8. Found
C 66.0; H 9.5; N 4.8.
1,12-Bis(4'-pentylpyridinium)dodec-6-yne dichloride (9) A solution of
diol 34 (15 mg, 0.07 mmol) in CH2Cl2 (2.5 mL) was cooled to 0 °C then
NEt3 (0.04 mL, 0.30 mmol), DMAP (4 mg, cat.) and p-TsCl (43 mg, 0.23
mmol) were added. The resulting solution was stirred under a nitrogen
atmosphere at 4 °C for 5 h, then poured onto a mixture of ice (8 g) and HCl
(1 M, 20 mL). The mixture was extracted with CH2Cl2 (3 x 20 mL) and the
combined organic phases were washed with sat. aq. NaHCO3 (40 mL) and
brine (40 mL), then dried (MgSO4), and the solvent removed under
reduced pressure. The residue was purified by flash chromatography
(Hex/EtOAc 4:0.3 → 4:0.6) and the required fractions were concentrated
to give tosylate 37 as a pale yellow liquid (23 mg, 60 %). 1H NMR (200 MHz,
CDCl3): δ 7.78 (4H, m), 7.34 (4H, m), 4.02 (4H, t, J = 6.5 Hz), 2.45 (6H, s),
2.09 (4H, m), 1.65 (4H, m), 1.41 (8H, m). Treatment of the ditosylate 37
(18 mg, 0.03 mmol) and 6 (12 mg, 0.08 mmol) according to the general
method for bispyridinium formation gave 9 as a brown oil (16 mg, 88 %).
1H NMR (200 MHz, CD3OD): δ 8.85 (4H, d, J = 6.4 Hz), 7.96 (4H, d, J =
6.4 Hz), 4.59 (4H, t, J = 7.4 Hz), 2.95 (4H, t, J = 7.4 Hz), 2.14 (4H, m), 2.03
(4H, m), 1.74 (4H, m), 1.51 – 1.35 (16H, m), 0.93 (6H, t, J = 6.7 Hz); 13C
NMR (50 MHz, CD3OD): δ 164.5, 144.2, 128.4, 79.9, 61.2, 35.7, 31.6, 31.1,
29.7, 28.7, 25.5, 22.6, 18.3, 13.4; m/z ESI (positive ion) 231 [M-2Cl-]2+
(100 %), 463 [M-2Cl--H+]+ (55), 497 [M-37Cl-]+ (100), 499 [M-35Cl-]+ (35).
1,10-Bis(4'-pentylpyridinium)dec-4,6-diyne dichloride (12) Treatment
of dec-4,6-diyne-1,10-diol (0.10 g, 0.58 mmol) according to the general
method for bromination gave 63 as a pale colourless solid (0.16 g, 94 %)
after flash chromatography (Hex/EtOAc 16:1
→
16:2.4). 1H NMR
(200 MHz, CDCl3): δ 3.51 (4H, t, J = 6.5 Hz), 2.46 (4H, t, J = 6.6 Hz), 2.05
(4H, m). Treatment of the dibromide 63 (0.16 g, 0.55 mmol) and 4-
pentylpyridine 6 (0.17 g, 1.20 mmol) according to the general method for
bispyridinium formation gave 12 as a dark yellow oil (0.11 g, 48 %).
1H NMR (300 MHz, CD3OD): δ 8.90 (4H, d, J = 6.8 Hz), 8.00 (4H, d, J =
6.3 Hz), 4.72 (4H, t, J = 7.8 Hz), 2.98 (4H, t, J = 6.5 Hz), 2.48 (4H, t, J =
6.5 Hz), 2.25 (4H, m), 1.76 (4H, m), 1.40 (8H, m), 0.93 (6H, t, J = 7.7 Hz);
13C NMR (75 MHz, CD3OD): δ 163.1, 142.9, 126.1, 74.0, 64.8, 58.7, 34.2,
30.0, 28.0, 20.9, 14.4, 11.6, 1 signal obscured or overlapping; m/z ESI
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800331
ChemMedChem
FULL PAPER
(positive ion) 215 [M-2Cl-]2+ (40 %), 430 [M-2Cl--H+]+ (100), 465 [M-37Cl-]+
(85), 467 [M-35Cl-]+ (15). Found [M-2Cl-]2+ 215.1670, [C30H42N2]2+ requires
215.1668. Anal. (C30H42Cl2N2·3.5H2O) calcd, C 63.8; H 8.7; N 5.0. Found
C 63.6; H 8.4; N 5.0.
59.3, 34.1, 29.9, 29.1, 28.0, 23.8, 20.1, 17.0, 11.7; m/z ESI (positive ion)
267 [M-2Cl-]2+ (100 %), 534 [M-2Cl--H+]+ (50), 569 [M-37Cl-]+ (30), 571 [M-
35Cl-]+ (15). Found [M-2Cl-]2+ 267.1975, [C38H50N2]2+ requires 267.1981.
Anal. (C38H50Cl2N2·3H2O) calcd, C 71.1; H 8.5; N 4.4. Found C 71.2; H 8.7;
N 4.3.
1,12-Bis(4'-pentylpyridinium)dodec-5,7-diyne
dichloride
(13)
Treatment of dodec-5,7-diyn-1,12-diol (0.10 g, 0.51 mmol) according to the
general method for bromination gave 64 as a pale yellow liquid (0.11 g,
70 %) after flash chromatography (Hex/EtOAc 4:1). 1H NMR (200 MHz,
CDCl3): δ 3.43 (4H, t, J = 6.5 Hz,), 2.32 (4H, t, J = 6.7 Hz), 1.99 (4H, m),
1.66 (4H, m); m/z APCI (positive ion) 195 [M+H]+. Treatment of the
dibromide 64 (61 mg, 0.19 mmol) and 6 (60 mg, 0.40 mmol) according to
the general method for bispyridinium formation gave 13 as a brown wax
(70 mg, 75 %). 1H NMR (300 MHz, CDCl3): δ 9.62 (4H, d, J = 6.0 Hz), 7.73
(4H, d, J = 6.0 Hz), 4.89 (4H, t, J = 7.6 Hz), 2.71 (4H, t, J = 7.3 Hz), 2.23
(4H, m), 2.07 (4H, m), 1.53 (8H, m), 1.18 (8H, m), 0.73 (6H, t, J = 6.8 Hz);
13C NMR (75 MHz, CDCl3): δ 163.1, 145.2, 128.3, 66.5, 60.2, 36.1, 31.4,
31.2, 29.5, 25.0, 22.5, 19.0, 14.1, 1 signal obscured or overlapping; m/z
ESI (positive ion) 229 [M-2Cl-]2+ (65 %), 458 [M-2Cl--H+]+ (100), 493 [M-
37Cl-]+ (90), 495 [M-35Cl-]+ (25). Found [M-2Cl-]2+ 229.1826, [C32H46N2]2+
requires 229.1825. Anal. (C32H46Cl2N2·5H2O) calcd, C 62.0; H 9.1; N 4.5.
Found C 61.8; H 8.8; N 4.7.
1,3-Bis[6'-(4''-pentylpyridinium)hexyl]benzene
dichloride
(17)
Treatment of 56 (0.08 g, 0.28 mmol) according to the general method for
bromination gave 67 as a pale yellow solid (0.11 g, 98 %) after flash
chromatography (Hex/EtOAc 16:1 → 16:2.4). 1H NMR (200 MHz, CDCl3):
δ 6.97 (4H, d, J = 6.0 Hz), 3.38 (4H, t, J = 6.6 Hz), 2.57 (4H, t, J = 7.8 Hz),
1.80 (4H, m), 1.52 (4H, m), 1.40 (8H, m). Treatment of the dibromide 67
(0.11 g, 0.29 mmol) and 6 (0.09 g, 0.61 mmol) according to the general
method for bispyridinium formation gave 17 as a dark yellow oil (0.10 g,
1
57 %). H NMR (300 MHz, CD3OD): δ 8.86 (4H, d, J = 6.6 Hz), 7.96 (4H,
d, J = 6.6 Hz), 7.10 (1H, t, J = 7.1 Hz), 6.95 (3H, m), 4.58 (4H, t, J = 7.6
Hz), 2.95 (4H, t, J = 7.4 Hz), 2.56 (4H, t, J = 7.4 Hz), 2.00 (4H, m), 1.75
(4H, m), 1.60 (4H, m), 1.40 (16H, m), 0.93 (6H, t, J = 6.5 Hz); 13C NMR
(75 MHz, CD3OD): δ 162.7, 142.6, 141.2, 127.1, 126.8, 122.7, 59.7, 34.2,
34.1, 30.0, 29.8, 28.2, 28.0, 27.2, 24.6, 20.9, 11.8, 1 signal obscured or
overlapping; m/z ESI (positive ion) 271 [M-2Cl-]2+ (95 %), 542 [M-2Cl--H+]+
(100), 577 [M-37Cl-]+ (70), 579 [M-35Cl-]+ (25). Found [M-2Cl-]2+ 271.2289,
[C38H58N2]2+ requires 271.2294. Anal. (C38H58Cl2N2·4H2O) calcd, C 66.5;
H 9.7; N 4.1. Found C 66.4; H 9.5; N 4.1.
1,4-Bis[6'-(4''-pentylpyridinium)-hex-1'-ynyl]benzene dichloride (14)
Treatment of 1,4-bis(6'-hydroxy-1'-hexynyl)benzene (0.22 g, 0.81 mmol)
according to the general method for bromination gave 65 as a light yellow
wax (0.28 g, 90 %) after flash chromatography Hex/EtOAc 16:1 → 16:2.4).
1H NMR (200 MHz, CDCl3): δ 7.29 (4H, s), 3.47 (4H, t, J = 7.0 Hz), 2.46
(4H, t, J = 6.5 Hz), 2.02 (4H, m), 1.77 (4H, m). Treatment of the dibromide
65 (0.12 g, 0.30 mmol) and 6 (0.09 g, 0.63 mmol) according to the general
method for bispyridinium formation gave 14 as a dark yellow oil (0.11 g,
1,2-Bis[6'-(4''-pentylpyridinium) hex-1'-ynyl]benzene dichloride (18)
Treatment of 49 (0.10 g, 0.37 mmol) according to the general method for
bromination gave 68 as a pale yellow solid (0.13 g, 90 %) after flash
chromatography (Hex/EtOAc 16:1 → 16:2.4). 1H NMR (200 MHz, CDCl3):
δ 7.36 (2H, m), 7.19 (2H, m), 3.48 (4H, t, J = 6.6 Hz), 2.53 (4H, t, J = 6.6
Hz), 2.06 (4H, m), 1.78 (4H, m). Treatment of the dibromide 68 (0.13 g,
0.34 mmol) and 6 (0.14 g, 0.35 mmol) according to the general method for
bispyridinium formation gave 18 as a dark yellow oil (0.09 g, 45 %).
1H NMR (300 MHz, CD3OD): δ 8.88 (4H, d, J = 6.5 Hz), 7.96 (4H, d, J =
6.5 Hz), 7.35 (4H, m), 4.67 (4H, t, J = 7.4 Hz), 2.96 (4H, t, J = 7.6 Hz), 2.58
(4H, m), 2.23 (4H, m), 1.68 (8H, m), 1.38 (8H, m), 0.93 (6H, t, J = 7.6 Hz);
13C NMR (75 MHz, CD3OD): δ 162.9, 142.6, 130.4, 127.4, 126.7, 124.7,
91.0, 78.9, 59.2, 34.0, 29.8, 29.0, 28.0, 23.8, 20.9, 17.1, 11.7; m/z ESI
(positive ion) 267 [M-2Cl-]2+ (53 %), 534 [M-2Cl--H+]+ (70), 569 [M-37Cl-]+
(100), 571 [M-35Cl-]+ (15). Found [M-2Cl-]2+ 267.1982, [C38H50N2]2+ requires
267.1981. Anal. (C38H50Cl2N2·3H2O) calcd, C 69.2; H 8.7; N 4.25. Found
C 69.0; H 8.8; N 4.1.
1
60 %). H NMR (300 MHz, CD3OD): δ 8.89 (4H, d, J = 6.4 Hz), 7.94 (4H,
d, J = 6.4 Hz), 7.26 (4H, s), 4.65 (4H, t, J = 7.5 Hz), 2.93 (4H, t, J = 7.3
Hz), 2.52 (4H, t, J = 7.0 Hz), 2.17 (4H, m), 1.71 (8H, m), 1.36 (8H, m), 0.90
(6H, t, J = 6.7 Hz); 13C NMR (75 MHz, CD3OD): δ 162.9, 142.7, 129.8,
126.8, 122.0, 89.2, 79.6, 59.3, 34.8, 30.0, 29.2, 28.0, 23.9, 21.0, 17.2,
2+
11.9; m/z ESI (positive ion) 232 [M-2Cl--C5H11
]
(100 %), 267 [M-2Cl-]2+
(50). Found [M-2Cl-]2+ 267.1983, [C38H50N2]2+ requires 267.1981. Anal.
(C38H50Cl2N2·4.5H2O) calcd, C 66.45; H 8.6; N 4.1. Found C 66.6; H 8.4;
N 4.0.
1,4-Bis[6'-(4''-pentylpyridinium)hexyl]benzene
dichloride
(15)
Treatment of 1,4-bis(6'-bromohexyl)benzene (0.16 g, 0.40 mmol) and 6
(0.12 g, 0.84 mmol) according to the general method for bispyridinium
formation gave 15 as a dark brown oil (0.14 g, 55 %). 1H NMR (300 MHz,
CD3OD): δ 8.84 (4H, d, J = 6.5 Hz), 7.95 (4H, d, J = 6.5 Hz), 7.06 (4H, s,
CH(2’)), 4.57 (4H, t, J = 7.6 Hz), 2.95 (4H, t, J = 7.3 Hz), 2.56 (4H, t, J =
7.0 Hz), 2.00 (4H, m), 1.75 (4H, m), 1.62 (4H, m), 1.41 (16H, m), 0.90 (6H,
t, J = 6.0 Hz); 13C NMR (75 MHz, CD3OD): δ 162.7, 142.6, 138.4, 126.8,
126.6, 59.7, 34.0, 33.7, 29.9, 29.8, 28.0, 27.1, 24.5, 20.9, 11.7, 1 signal
obscured or overlapping; m/z ESI (positive ion) 271 [M-2Cl-]2+ (100 %), 542
[M-2Cl--H+]+ (85), 577 [M-37Cl-]+ (50), 579 [M-35Cl-]+ (20). Found [M-2Cl-]2+
271.2289, [C38H58N2]2+ requires 271.2294. Anal. (C38H58Cl2N2·3.5H2O)
calcd, C 67.4; H 9.6; N 4.1. Found C 67.5; H 9.5; N 4.1.
1,2-Bis[6'-(4''-pentylpyridinium)hexyl]benzene
dichloride
(19)
Treatment of 57 (0.10 g, 0.37 mmol) according to the general method for
bromination gave 69 as a pale yellow solid (0.13 g, 90 %) after flash
chromatography (Hex/EtOAc 16:1 → 16:2.4). 1H NMR (200 MHz, CDCl3):
δ 7.12 (4H, s), 3.41 (4H, t, J = 6.8 Hz), 2.60 (4H, t, J = 7.3 Hz), 1.85 (4H,
m), 1.52 (4H, m), 1.49 (8H, m). Treatment of the dibromide 69 (0.13 g,
0.35 mmol) and 6 (0.13 g, 0.33 mmol) according to the general method for
bispyridinium formation gave 19 as a pale brown oil (0.08 g, 43 %).
1H NMR (200 MHz, CD3OD): δ 8.80 (4H, d, J = 6.6 Hz), 7.93 (4H, d, J =
6.6 Hz), 7.07 (4H, m), 4.57 (4H, t, J = 7.6 Hz), 2.94 (4H, t, J = 7.4 Hz), 2.59
(4H, t, J = 7.4 Hz), 1.98 (4H, m), 1.63 (8H, m), 1.39 (16H, m), 0.92 (6H, t,
J = 7.4 Hz); 13C NMR (100 MHz, CD3OD): δ 165.4, 145.0, 141.1, 130.3,
129.2, 127.0, 62.2, 36.5, 33.4, 32.4, 32.2, 30.5, 30.0, 27.2, 23.4, 14.2, 1
signal obscured or overlapping; m/z ESI (positive ion) 271 [M-2Cl-]2+
(35 %), 542 [M-2Cl--H+]+ (100), 577 [M-37Cl-]+ (70), 579 [M-35Cl-]+ (20).
Found [M-2Cl-]2+ 271.2289, [C19H29N]2+ requires 271.2294. Anal.
(C38H58Cl2N2·2H2O) calcd, C 68.3; H 9.7; N 4.2. Found C 67.9; H 9.6; N
4.1.
1,3-Bis[6'-(4''-pentylpyridinium) hex-1'-ynyl]benzene dichloride (16)
Treatment of 48 (0.14 g, 0.52 mmol) according to the general method for
bromination gave 66 as a pale yellow solid (0.15 g, 72 %) after flash
chromatography (Hex/EtOAc 16:1 → 16:2.4). 1H NMR (300 MHz, CDCl3):
δ 7.42 (1H, s), 7.30 (2H, d, J = 6.0 Hz), 7.18 (1H, t, J = 7.0 Hz), 3.47 (4H,
t, J = 7.0 Hz), 2.45 (4H, t, J = 7.5 Hz), 2.05 (4H, m), 1.74 (4H, m).
Treatment of the dibromide 66 (0.07 g, 0.18 mmol) and 6 (0.06 g, 0.39
mmol) according to the general method for bispyridinium formation gave
16 as a pale brown wax (0.05 g, 50 %). 1H NMR (300 MHz, CD3OD): δ
8.83 (4H, d, J = 6.5 Hz), 7.92 (4H, d, J = 6.5 Hz), 7.24 (1H, s), 7.22 (3H,
m), 4.61 (4H, m), 2.90 (4H, t, J = 7.5 Hz), 2.48 (4H, t, J = 7.0 Hz), 2.18 (4H,
m), 1.70 (8H, m), 1.35 (8H, m), 0.89 (6H, t, J = 6.7 Hz); 13C NMR (75 MHz,
CD3OD): δ 162.9, 142.6, 132.7, 130.0, 127.4, 127.1, 122.8, 88.0, 79.0,
1,4-Bis[5'-(4''-pentylpyridinium) pent-1'-ynyl]benzene dichloride (20)
Treatment of 54 (0.10 g, 0.42 mmol) according to the general method for
bromination gave 70 as a pale colourless solid (0.14 g, 90 %) after flash
chromatography (Hex/EtOAc 16:1 → 16:2.4). 1H NMR (200 MHz, CDCl3):
δ 7.29 (4H, s), 3.56 (4H, t, J = 7.0 Hz), 2.60 (4H, t, J = 6.5 Hz), 2.12 (4H,
m). Treatment of the dibromide 70 (0.14 g, 0.39 mmol) and 6 (0.14 g, 0.31
10
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800331
ChemMedChem
FULL PAPER
mmol) according to the general method for bispyridinium formation gave
20 as a dark yellow oil (0.07 g, 40 %). 1H NMR (300 MHz, CD3OD): δ 8.88
(4H, d, J = 6.5 Hz), 7.92 (4H, d, J = 6.4 Hz), 7.26 (4H, s), 4.74 (4H, t, J =
7.5 Hz), 2.90 (4H, t, J = 7.4 Hz), 2.63 (4H, t, J = 7.5 Hz), 2.37 (4H, m), 1.65
(4H, m), 1.36 (8H, m), 0.92 (6H, t, J = 7.7 Hz); 13C NMR (75 MHz, CD3OD):
δ 164.6, 144.4, 131.6, 128.1, 123.0, 89.0, 81.0, 60.6, 35.5, 31.4, 29.6, 29.3,
22.4, 16.1, 13.2; m/z ESI (positive ion) 253 [M-2Cl-]2+ (100 %), 506 [M-2Cl-
-H+]+ (60). Found [M-2Cl-]2+ 253.1820, [C36H46N2]2+ requires 253.1825.
Anal. (C36H46Cl2N2·5.5H2O) calcd, C 63.9; H 8.4; N 4.0. Found C 63.7; H
8.2; N 4.0.
chromatography (2 x Hex/EtOAc 16:1 → 4:1) and the required fractions
were concentrated to give 73 as a pale yellow wax (52 mg, 74 %). 1H NMR
(300 MHz, CDCl3): δ 3.40 (4H, t, J = 6.8 Hz), 2.69 (4H, t, J = 7.4 Hz), 1.87
(4H, m), 1.61 (4H, m), 1.41 (8H, m). Treatment of the dibromide 73 (52
mg, 0.11 mmol) and 6 (34 mg, 0.23 mmol) according to the general method
for bispyridinium formation gave 23 as a dark yellow wax (80 mg, 73 %).
1H NMR (200 MHz, CD3OD): δ 8.90 (4H, d, J = 6.6 Hz), 7.98 (4H, d, J =
6.6 Hz), 4.62 (4H, t, J = 7.4 Hz), 2.97 (4H, t, J = 7.5 Hz), 2.70 (4H, t, J =
6.6 Hz), 2.02 (4H, m), 1.77 (4H, m), 1.62 (4H, m), 1.41 (16H, m), 0.93 (6H,
t, J = 7.0 Hz); 13C NMR (75 MHz, CD3OD): δ 164.2, 144.1, 128.6, 61.1,
35.6, 31.4, 31.3, 29.5, 29.1, 28.6, 25.6, 22.4, 22.3, 13.3, not all aromatic
signals were observed due to C-F coupling; m/z ESI (positive ion) 307 [M-
2Cl-]2+ (100 %), 613 [M-2Cl--H+]+ (50), 649 [M-37Cl-]+ (95), 651 [M-35Cl-]+
(35). Found [M-2Cl-]2+ 307.2097, [C38H54N2F4]2+ requires 307.2106. Anal.
(C38H54Cl2N2F4·3H2O) calcd, C 61.7; H 8.2; N 3.8. Found C 61.6; H 8.1; N
3.7.
1,4-Bis[5''-(4'-pentylpyridinium)pentyl]benzene
dichloride
(21)
Treatment of 62 (0.11 g, 0.46 mmol) according to the general method for
bromination gave 71 as a pale yellow solid (0.16 g, 92 %) after flash
chromatography (Hex/EtOAc 16:1 → 16:2.4). 1H NMR (300 MHz, CDCl3):
δ 7.08 (4H, s), 3.40 (4H, t, J = 6.8 Hz), 2.59 (4H, t, J = 7.5 Hz), 1.89 (4H,
m), 1.60 (4H, m), 1.50 (4H, m). Treatment of the dibromide 71 (0.16 g,
0.42 mmol) and 6 (0.13 g, 0.88 mmol) according to the general method for
bispyridinium formation gave 21 as a pale brown wax (0.08 g, 41 %).
1H NMR (300 MHz, CD3OD): δ 8.81 (4H, d, J = 6.5 Hz), 7.93 (4H, d, J =
6.5 Hz), 7.07 (4H, s), 4.55 (4H, t, J = 7.6 Hz), 2.93 (4H, t, J = 7.3 Hz), 2.57
(4H, t, J = 7.0 Hz), 2.03 (4H, m), 1.68 (8H, m), 1.39 (12H, m), 0.93 (6H, t,
J = 7.7 Hz); 13C NMR (75 MHz, CD3OD): δ 162.7, 142.5, 138.2, 126.9,
126.6, 59.6, 34.0, 33.5, 29.9, 29.6, 29.4, 28.0, 24.1, 20.9, 11.7; m/z ESI
(positive ion) 257 [M-2Cl-]2+ (95 %), 514 [M-2Cl--H+]+ (100), 549 [M-37Cl-]+
(80), 551 [M-35Cl-]+ (25). Found [M-2Cl-]2+ 257.2133, [C36H54N2]2+ requires
257.2138. Anal. (C36H54Cl2N2·4H2O) calcd, C 65.7; H 9.4; N 4.3. Found C
65.65; H 9.2; N 4.2.
2,5-Bis[6'-(4''-pentylpyridinium) hex-1'-ynyl]thiophene dichloride (24)
A solution of 51 (0.07 g, 0.28 mmol) in CH2Cl2 (8 mL) was cooled to 0 °C
then NEt3 (0.10 mL, 0.72 mmol), DMAP (4 mg, cat.) and p-TsCl (0.11 g,
0.61 mmol) were added. The resulting solution was stirred under a nitrogen
atmosphere at 4 °C for 7 h, then poured onto a mixture of ice (8 g) and HCl
(1 M, 20 mL). The mixture was extracted with CH2Cl2 (3 x 20 mL) and the
combined organic phases were washed with sat. aq. NaHCO3 (40 mL) and
brine (40 mL), then dried (MgSO4), and the solvent removed under
reduced pressure. The residue was further purified by flash
chromatography (Hex/EtOAc 4:0.3 → 4:0.6) and the required fractions
were concentrated to give the tosylate 74 as a brown oil (0.11 g, 70 %).
1H NMR (200 MHz, CDCl3): δ 7.77 (4H, d, J = 6.6 Hz), 7.32 (4H, d, J = 6.6
Hz), 6.89 (2H, s), 4.08 (4H, t, J = 6.0 Hz), 2.43 (6H, s), 2.38 (4H, t, J = 6.1
Hz), 1.79 (4H, m), 1.63 (4H, m); 13C NMR (200 MHz, CDCl3): δ 145.4,
133.6, 131.5, 130.5, 128.4, 124.6, 94.3, 74.8, 70.5, 28.6, 24.9, 22.2, 19.6.
Treatment of the ditosylate 74 (93 mg, 0.19 mmol) and 6 (56 mg, 0.37
mmol) according to the general method for bispyridinium formation gave
24 as a dark yellow oil (68 mg, 70 %). 1H NMR (300 MHz, CD3OD): δ 8.90
(4H, d, J = 6.4 Hz), 7.97 (4H, d, J = 6.4 Hz), 6.95 (2H, s), 4.66 (4H, t, J =
7.5 Hz), 2.95 (4H, t, J = 7.3 Hz), 2.55 (4H, t, J = 7.0 Hz), 2.16 (4H, m), 1.71
(8H, m), 1.37 (8H, m), 0.92 (6H, t, J = 7.7 Hz); 13C NMR (75 MHz, CD3OD):
δ 163.4, 143.7, 130.4, 128.9, 124.3, 94.9, 73.9, 60.7, 35.6, 31.4, 30.4, 29.5,
25.2, 22.4, 18.8, 13.3; m/z ESI (positive ion) 270 [M-2Cl-]2+ (100 %), 540
[M-2Cl--H+]+ (25), 575 [M-37Cl-]+ (45), 577 [M-35Cl-]+ (18). Found [M-2Cl-]2+
270.1765, [C36H48N2S]2+ requires 270.1764. Anal. (C36H48Cl2N2S·3.5H2O)
calcd, C 64.1; H 8.1; N 4.15. Found C 64.4; H 7.9; N 4.2.
1,4-Bis[6'-(4''-pentylpyridinium)
hex-1'-ynyl]-2,3,5,6-
tetrafluorobenzene dichloride (22) Carbon tetrabromide (116 mg, 0.35
mmol) was added to a solution of 50 (46 mg, 0.13 mmol) in dry CH2Cl2 (12
mL) and the resulting mixture was stirred at RT for 10 min.
Triphenylphosphine (92 mg, 0.35 mmol) in dry CH2Cl2 (4 mL) was added
to the solution and the mixture was stirred for 24 h at 45 °C under a
nitrogen atmosphere. The solvent was removed under reduced pressure
then Hex/EtOAc (1:1, 20 mL) was added to the residue. The resulting
solution was washed with NaHCO3 (2 x 30 mL) and brine (30 mL), dried
(MgSO4), and the solvent was removed under reduced pressure. The
residue was triturated with hexane then the filtrate was concentrated and
removed under reduced pressure. The residue was purified by flash
chromatography (Hex/EtOAc 16:1 → 4:1) and the required fractions were
concentrated to give 72 as a yellow oil (64 mg, 80 %). 1H NMR (200 MHz,
CDCl3): δ 3.47 (4H, t, J = 7.0 Hz), 2.57 (4H, t, J = 6.5 Hz), 2.06 (4H, m),
1.81 (4H, m). Treatment of the dibromide 72 (65 mg, 0.14 mmol) and 6
(45 mg, 0.30 mmol) according to the general method for bispyridinium
formation gave 22 as a pale brown wax (66 mg, 75 %). 1H NMR (200 MHz,
CD3OD): δ 8.87 (4H, d, J = 6.4 Hz), 7.99 (4H, d, J = 6.4 Hz), 4.68 (4H, t, J
= 7.0 Hz), 2.96 (4H, t, J = 7.4 Hz), 2.56 (4H, t, J = 7.5 Hz), 2.20 (4H, m),
1.75 (8H, m), 1.40 (8H, m), 0.94 (6H, t, J = 7.7 Hz); 13C NMR (50 MHz,
CD3OD): δ 164.7, 144.2, 128.4, 66.5, 60.8, 35.7, 31.5, 30.6, 29.7, 25.0,
22.5, 19.0, 13.3, not all aromatic signals were observed due to C-F
coupling; m/z ESI (positive ion) 303 [M-2Cl-]2+ (100 %), 606 [M-2Cl--H+]+
(40), 641 [M-37Cl-]+ (45), 643 [M-35Cl-]+ (15). Found [M-2Cl-]2+ 303.1794,
[C38H46N2F4]2+ requires 303.1793. Anal. (C38H46Cl2N2F4·3H2O) calcd, C
62.4; H 7.2; N 3.8. Found C 62.5; H 7.5; N 3.7.
2,5-Bis[6'-(4''-pentylpyridinium)hexyl]thiophene dichloride (25)
A
solution of 59 (0.05 g, 0.20 mmol) in CH2Cl2 (7 mL) was cooled to 0 °C
then NEt3 (0.07 mL, 0.53 mmol), DMAP (3 mg, cat.) and p-TsCl (0.09 g,
0.45 mmol) were added. The resulting solution was stirred under a nitrogen
atmosphere at 4 °C for 10 h, then poured onto a mixture of ice (8 g) and
HCl (1 M, 20 mL). The mixture was extracted with CH2Cl2 (3 x 20 mL) and
the combined organic phases were washed with sat. aq. NaHCO3 (40 mL)
and brine (40 mL), then dried (MgSO4), and the solvent removed under
reduced pressure. The residue was further purified by flash
chromatography (Hex/EtOAc 4:0.3 → 4:0.6) and the required fractions
were concentrated to give the tosylate 75 (0.12 g, 56 %). 1H NMR
(200 MHz, CDCl3): δ 7.78 (4H, d, J = 6.3 Hz), 7.34 (4H, d, J = 6.5 Hz), 6.52
(4H, s), 4.02 (4H, t, J = 7.2 Hz), 2.69 (4H, t, J = 7.5 Hz), 2.44 (6H, s), 1.63
(8H, m), 1.30 (8H, m). Treatment of the ditosylate 75 (90 mg, 0.15 mmol)
1,4-Bis[6'-(4''-pentylpyridinium)hexyl]-2,3,5,6-tetrafluorobenzene
dichloride (23) Carbon tetrabromide (95 mg, 0.28 mmol) was added to a
solution of 56 (40 mg, 0.11 mmol) in dry CH2Cl2 (5 mL) and the resulting
solution was stirred at RT for 10 min. Triphenylphosphine (75 mg, 0.28
mmol) in dry CH2Cl2 (2 mL) was added then mixture was stirred for 4 h at
45 °C under a nitrogen atmosphere. The solvent was removed under
reduced pressure then Hex/EtOAc (1:1, 20 mL) was added to the residue,
washed with NaHCO3 (2 x 30 mL) and brine (30 mL), dried (MgSO4), and
the solvent was removed under reduced pressure. The residue was
triturated with hexane and the filtrate was collected, and the solvent was
removed under reduced pressure. The residue was purified by flash
and
6 (50 mg, 0.33 mmol) according to the general method for
bispyridinium formation gave 25 as a pale brown oil (67 mg, 72 %).
1H NMR (300 MHz, CD3OD): δ 8.85 (4H, d, J = 6.5 Hz), 7.96 (4H, d, J =
6.5 Hz), 6.54 (4H, s), 4.58 (4H, t, J = 7.6 Hz), 2.96 (4H, t, J = 7.3 Hz), 2.74
(4H, t, J = 7.0 Hz), 2.01 (4H, m), 1.78 (4H, m), 1.65 (4H, m), 1.41 (16H, m),
0.93 (6H, t, J = 7.7 Hz); 13C NMR (75 MHz, CD3OD): δ 162.7, 142.6, 141.4,
126.7, 123.3, 59.7, 34.1, 30.1, 29.9, 29.8, 28.3, 28.0, 26.9, 24.4, 20.9,
11.8; m/z ESI (positive ion) 274 [M-2Cl-]2+ (50 %), 548 [M-2Cl--H+]+ (45),
583 [M-37Cl-]+ (100), 585 [M-35Cl-]+ (40). Found [M-2Cl-]2+ 274.2078,
[C36H56N2S]2+ requires 274.2077. Anal. (C36H56Cl2N2S·4.5H2O) calcd, C
61.6; H 9.3; N 4.0. Found C 61.5; H 9.1; N 4.0.
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800331
ChemMedChem
FULL PAPER
4,4'-Bis[5''-(4'''-pentylpyridinium) pent-1''-ynyl]biphenyl dichloride
(26) Carbon tetrabromide (0.59 g, 1.77 mmol) was added to a solution of
52 (0.22 g, 0.81 mmol) in dry CH2Cl2 (8 mL) and the resulting solution was
stirred at RT for 10 min. Triphenylphosphine (0.46 g, 1.77 mmol) in dry
CH2Cl2 (4 mL) was added then the mixture was stirred for 9 h at 50 °C
under a nitrogen atmosphere. The solvent was removed under reduced
pressure then Hex/EtOAc (1:1, 20 mL) was added to the residue. The
resulting solution was washed with NaHCO3 (2 x 30 mL) and brine (30 mL),
dried (MgSO4) and the solvent was removed under reduced pressure. The
residue was triturated with hexane then filtrates were collected, and the
solvent was removed under reduced pressure. The residue was purified
by flash chromatography (Hex/EtOAc 4:0.3 → 4:0.6), and the required
fractions were concentrated to give 76 as a yellow wax (80 mg, 36 %).
1H NMR (200 MHz, CDCl3): δ 7.48 (8H, m), 3.60 (4H, t, J = 6.5 Hz), 2.64
(4H, t, J = 6.7 Hz), 2.17 (4H, m). Treatment of the dibromide 76 (6 mg, 0.01
mmol) and 6 (5 mg, 0.03 mmol) according to the general method for
bispyridinium formation gave 26 as a dark yellow oil (6 mg, 76%). 1H NMR
(300 MHz, CD3OD): δ 8.90 (4H, d, J = 6.6 Hz), 7.92 (4H, d, J = 6.6 Hz),
7.59 (4H, d, J = 6.0 Hz), 7.37 (4H, d, J = 6.0 Hz), 4.77 (4H, t, J = 7.0 Hz),
2.80 (4H, t, J = 7.5 Hz), 2.66 (4H, t, J = 6.5 Hz), 2.34 (4H, m), 1.61 (4H,
m), 1.31 (8H, m), 0.86 (6H, t, J = 6.8 Hz); 13C NMR (75 MHz, CD3OD): δ
164.6, 144.4, 139.9, 132.2, 128.1, 126.8, 123.0, 88.2, 81.7, 70.7, 35.5,
31.4, 29.5, 29.2, 22.3, 16.2, 13.2; m/z ESI (positive ion) 291 [M-2Cl-]2+
(100 %), 582 [M-2Cl--H+]+ (25). Found [M-2Cl-]2+ 291.1972, [C42H50N2]2+
requires 291.1981. Anal. (C42H50Cl2N2·4H2O) calcd, C 69.5; H 8.1 N 3.9.
Found C 69.2; H 8.3; N 4.1.
(4H, t, J = 6.5 Hz), 2.64 (4H, t, J = 6.7 Hz), 2.17 (4H, m). Treatment of the
dibromide 78 (30 mg, 0.06 mmol) and 6 (22 mg, 0.14 mmol) according to
the general method for bispyridinium formation gave 28 as a pale brown
wax (37 mg, 85 %). 1H NMR (300 MHz, CD3OD): δ 8.95 (4H, d, J = 6.0
Hz), 7.91 (4H, d, J = 6.5 Hz), 7.74 (2H, d, J = 6.5 Hz), 7.50 (2H, s), 7.40
(2H, d, J = 6.7 Hz), 4.78 (4H, t, J = 7.0 Hz), 2.71 (8H, m), 3.87 (2H, s), 2.37
(4H, m), 1.52 (4H, m), 1.23 (8H, m), 0.81 (6H, t, J = 7.7 Hz); 13C NMR
(75 MHz, CD3OD): δ 164.5, 144.4, 143.9, 141.1, 130.7, 128.2, 128.1,
122.2, 121.3, 87.5, 82.5, 60.8, 36.3, 35.5, 31.5, 29.6, 29.0, 22.3, 16.3,
13.2; m/z ESI (positive ion) 297 [M-2Cl-]2+ (100 %), 593 [M-2Cl--H+]+ (100).
Found [M-2Cl-]2+ 297.1973, [C43H50N2]2+ requires 297.1981. Anal.
(C43H50Cl2N2·5H2O) calcd, C 68.3; H 7.9; N 3.7. Found C 68.3; H 7.6; N
3.8.
2,7-Bis[5'-(4''-pentylpyridium)pentyl]fluorene dichloride (29) NEt3
(0.03 mL, 0.23 mmol), DMAP (3 mg, cat.) and p-TsCl (0.03 g, 0.17 mmol)
were added to a solution of 61 (0.02 g, 0.06 mmol) in CH2Cl2/DMF (4 mL,
1:1). The resulting solution was stirred under a nitrogen atmosphere at RT
for 24 h. The mixture was diluted with hexane (20 mL) and washed with
H2O (4 x 20 mL) and HCl (1 M, 2 x 20 mL) then the organic phase was
died (MgSO4), and the solvent removed under reduced pressure. The
residue was purified by flash chromatography (Hex/EtOAc 4:0.3 → 4:0.6)
and the required fractions were concentrated to give the tosylate 79 (0.11
g, 70 %). 1H NMR (300 MHz, CDCl3): δ 7.77 (4H, d, J = 6.5 Hz), 7.63 (2H,
d, J = 6.5 Hz), 7.30 (6H, m), 7.15 (2H, d, J = 6.5 Hz), 4.02 (4H, t, J = 6.5
Hz), 3.82 (2H, s), 2.62 (4H, t, J = 6.5 Hz), 2.42 (6H, s), 1.64 (4H, m), 1.37
(8H, m). Treatment of the ditosylate 79 (27 mg, 0.04 mmol) and 6 (14 mg,
0.09 mmol) according to the general method for bispyridinium formation
4,4'-Bis[5''-(4'''-pentylpyridinium)pentyl]biphenyl
dichloride
(27)
1
Carbon tetrabromide (0.59 g, 1.77 mmol) was added to a solution of 60
(0.22 g, 0.81 mmol) in dry CH2Cl2 (8 mL) and the resulting solution was
stirred at RT for 10 min. Triphenylphosphine (0.46 g, 1.77 mmol) in dry
CH2Cl2 (4 mL) was added and the mixture was stirred for 9 h at 50 °C
under a nitrogen atmosphere. The solvent was removed under reduced
pressure and Hex/EtOAc (1:1, 20 mL) was added to the residue. The
resulting solution was washed with NaHCO3 (2 x 30 mL) and brine (30 mL),
dried (MgSO4) and the solvent was removed under reduced pressure. The
residue was triturated with hexane then filtrates were collected, and the
solvent was removed under reduced pressure. The residue was purified
by flash chromatography (Hex/EtOAc 4:0.3 → 4:0.6), and the required
fractions were concentrated to give 77 (0.10 g, 95 %). 1H NMR (300 MHz,
CDCl3): δ 7.52 (4H, m), 7.25 (4H, m), 3.44 (4H, t, J = 6.8 Hz), 2.68 (4H, t,
J = 7.5 Hz), 1.93 (4H, m), 1.61 (8H, m). Treatment of the dibromide 77
(105 mg, 0.23 mmol) and 6 (73 mg, 0.49 mmol) according to the general
method for bispyridinium formation gave 27 as a dark yellow oil (95 mg,
gave 29 as a dark yellow oil (25 mg, 90 %). H NMR (300 MHz, CD3OD):
δ 8.75 (4H, d, J = 6.4 Hz), 7.86 (4H, d, J = 6.4 Hz), 7.65 (2H, d, J = 6.0
Hz), 7.33 (2H, s), 7.13 (2H, d, J = 6.1 Hz), 4.55 (4H, t, J = 7.3 Hz), 3.77
(2H, s), 2.89 (4H, t, J = 7.5 Hz), 2.71 (4H, t, J = 7.5 Hz), 2.06 (4H, m), 1.75
(8H, m), 1.33 (12H, m), 0.92 (6H, t, J = 7.7 Hz); 13C NMR (300 MHz,
CD3OD): δ 164.2, 144.0, 143.7, 140.8, 139.8, 128.0, 127.3, 125.2, 119.4,
60.9, 36.4, 35.4, 31.4, 30.9, 30.7, 29.4, 25.1, 22.3, 13.2, 1 signal obscured
or overlapping; m/z ESI (positive ion) 301 [M-2Cl-]2+ (100 %), 602 [M-2Cl--
H+]+ (18), 637 [M-37Cl-]+ (20), 639 [M-35Cl-]+ (10). Found [M-2Cl-]2+
.
301.2294, [C43H58N2]2+ requires 301.2285. Anal. (C43H58Cl2N2 0.5H2O)
calcd, C 75.4; H 8.7; N 4.1. Found C 75.2; H 9.2; N 3.9.
Fungal isolates and media. A virulent clinical isolate of C. neoformans
var. grubii (serotype A), H99, was kindly supplied by Dr. Gary Cox (Duke
University Medical Center, Durham, NC, USA) and subcultured onto
Sabouraud dextrose agar (SDA) at 30°C. H99 secretes high levels of
phospholipase B activity attributable to the phospholipase B1 (PLB1)
enzyme, which was used for the phospholipase inhibition assays. The
PLB1 deletion mutant (Δplb1) and reconstituted strain (Δplb1::REC15)
were created from strain H99 as described in Cox et al. 9 The Δsec14-1
deletion mutant was also created from strain H99 as described in
Chayakulkeeree et al. 31
1
80 %). H NMR (300 MHz, CD3OD): δ 8.80 (4H, d, J = 6.5 Hz), 7.89 (4H,
d, J = 6.5 Hz), 7.50 (4H, d, J = 6.0 Hz), 7.24 (4H, d, J = 6.4 Hz), 4.57 (4H,
t, J = 7.6 Hz), 2.88 (4H, t, J = 7.3 Hz), 2.67 (4H, t, J = 7.0 Hz), 2.04 (4H,
m), 1.75 (8H, m), 1.33 (12H, m), 0.90 (6H, t, J = 6.5 Hz); 13C NMR (75 MHz,
CD3OD): δ 164.4, 144.2, 141.5, 138.9, 129.3, 128.3, 126.8, 61.2, 35.7,
35.1, 31.6, 31.1, 30.8, 29.7, 25.5, 22.5, 13.4; m/z ESI (positive ion) 295
[M-2Cl-]2+ (100 %), 589 [M-2Cl--H+]+ (25), 625 [M-37Cl-]+ (24), 627 [M-35Cl-]+
(10). Found [M-2Cl-]2+ 295.2285, [C42H58N2]2+ requires 295.2294. Anal.
(C42H58Cl2N2·2.5H2O) calcd, C 71.5; H 8.9; N 4.0. Found C 71.8; H 8.9; N
3.95.
Antifungal susceptibility testing. The antifungal activity of the
compounds was measured using the standard broth microdilution methods
of the US National Committee for Clinical Laboratory Standards (NCCLS)
for yeasts26 and filamentous fungi.27 All compounds were preparedas stock
solutions (generally 700 µM; in some cases lower concentrations were
used) in water and diluted in RPMI medium according to NCCLS
methodology. Two drug-free controls were included in each test run, one
contained medium alone (sterile control) and the other, medium plus the
inoculum (growth control). The minimal inhibitory concentration (MIC) was
defined as that which produced no visible growth after 48, or 72 hours, of
culture at 35 oC.26,27 All tests were performed in duplicate in at least two
independent experiments and the MICs were within ± one dilution step in
all cases (values are given as single number where all four replicates were
identical and as a range when replicates varied).
2,7-Bis[5'-(4''-pentylpyridium)pent-1'-ynyl]fluorene dichloride (28)
Carbon tetrabromide (0.59 g, 1.77 mmol) was added as to a solution of 53
(0.22 g, 0.81 mmol) in dry CH2Cl2 (8 mL) and stirred at RT for 10 min.
Triphenylphosphine (0.46 g, 1.77 mmol) in dry CH2Cl2 (4 mL) was added
to the solution and the mixture was stirred for 9 h at 50 °C under a nitrogen
atmosphere. The solvent was removed under reduced pressure then
Hex/EtOAc (1:1, 20 mL) was added to the residue. The resulting solution
was washed with NaHCO3 (2 x 30 mL) and brine (30 mL), dried (MgSO4)
and the solvent was removed under reduced pressure. The residue was
triturated with hexane then filtrates were collected, and the solvent was
removed under reduced pressure. The residue was purified by flash
chromatography (Hex/EtOAc 4:0.3 → 4:0.6) the required fractions were
concentrated to give 78 (30 mg, 44 %). 1H NMR (200 MHz, CDCl3): δ 7.66
(2H, d, J = 6.5 Hz), 7.56 (2H, s), 7.41 (2H, d, J = 6.5 Hz), 3.84 (2H, s), 3.61
Haemolytic activity assay. Human blood was collected in 10 mL
Vacutainer tubes containing potassium-EDTA as anticoagulant. The blood
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201800331
ChemMedChem
FULL PAPER
from each Vacutainer was transferred to a 50 mL centrifuge tube and the
cells washed three times with 30 mL of calcium- and magnesium-free
phosphate-buffered saline (PBS; Gibco). Cells were collected by
centrifugation at 2,000 × g for 10 mins in a Beckman TJ-6 centrifuge. The
third supernatant was clear and colourless. Cells were stored in PBS (20
mL) for up to two weeks. Haemolysis was assessed by mixing 0.5 mL of
the cell suspension with 0.5 ml of test substance from stock solutions of
700, 350, 175, 70 and 7 µM in PBS (final erythrocyte concentration around
0.5 × 109 per mL). The mixtures were incubated with gentle shaking at 37
ºC for 1 h, centrifuged at 2,000 × g for 10 mins, the supernatant diluted 10-
fold with PBS, and the optical density measured at 540 nm. The values for
0% and 100% lysis were determined by incubating cells with PBS or 0.1 %
(w/v) Triton X-100 (in water), respectively. Assays were carried out in
triplicate and the difference between runs was < 5% in all cases. The
concentration of test compounds in the assays was 350, 175, 87.5, 35 and
All reactions were terminated by adding 0.5 mL of chloroform:methanol
(2:1 v/v). The reaction products were extracted by the method of Bligh and
Dyer,41 separated by TLC using chloroform: methanol:water (65:25:4;
v/v/v) as the eluting solvent, detected by iodine staining and quantified by
beta counting as previously described.40
Testing of bis(alkylpyridinium)s as inhibitors of fungal PLB1.
Solutions of bis(alkylpyridinium)s were prepared as stock solutions of 700
μM in assay buffer containing 5 mM EDTA, which was then diluted serially
with buffer to give solutions with concentrations of 70, 7 and 0.7 μM. In
each assay, 45 µL of the stock or diluted solutions was used, and the final
volume of 125 μL was made up of substrate, enzyme and buffer. This gave
solutions with compound concentrations of 250, 25, 2.5 and 0.25 M. The
radiometric assay was carried out as above. Inhibition was calculated as
the percent of substrates (DPPC or Lyso-PC) remaining in the case of PLB
and LPL activities, respectively, or of DPPC produced, in the case of the
LPTA activity, measured against inhibitor-free controls, the results of which
had been normalised to 100 %. All assays were performed in triplicate; the
variation between runs was < 10 % in all cases. Compounds which showed
3.5 µM. The haemolytic concentration, HC50
, was defined as the
concentration at which there was 50% lysis determined by plotting % lysis
against concentration of drug.
Cytotoxicity. Cytotoxicity was assessed in a human lung epithelial cell
line (A549) and in Madin-Darby canine kidney (MDCK) cells which were
maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, GIBCO)
supplemented with 10% fetal bovine serum (FBS). 3 x 104 cells were
seeded onto each well of a 96 well plate, and incubated in supplemented
DMEM at 37o C for 24 h. 50 µL of the supernatant medium was removed
and replaced with 50 µL of test compound at various concentrations. All
drugs were prepared as stock solutions of 700 µM in DMEM. The mixture
was incubated for 3 h at 37o C. Negative and positive controls contained
equivalent volumes of DMEM and 0.1% (w/v) Triton X-100 in DMEM,
respectively. Cell viability was determined by the MTS tetrazolium
inhibition were then assayed over a narrower concentration range to
42
determine the IC50
.
Porcine pancreatic PLA2 activity. Porcine pancreatic phospholipase A2
was suspended in 3.2 M ammonium sulfate (2.9 mg protein/mL, Sigma
St.Louis MO, USA). One part of well-mixed enzyme suspension was
added to 4 parts of buffer [10 mM Tris/HCl, 10 mM CaCl2, pH 8.2].30 Activity
and inhibition by test compounds were then measured by the radiometric
method described above, except that 25 μL of enzyme solution was used,
and the reaction time was 1 hour. These conditions resulted in ~ 60%
substrate conversion in the inhibitor-free control, which was normalised to
100% and inhibition in the presence of compounds calculated against it.
All assays were performed in triplicate; the variation between runs was <
method28 and the dose at which cell viability was reduced by 50% (CC50
)
is reported (values determined by plotting % viability against concentration
of drug (tested at 350, 175, 87.5, 44, 17.5 and 3.5 µM). Assays were
carried out in triplicate and mean values ± SD are reported. Data analysis
was performed using Graphpad Prism software.41
10 % in all cases. Compounds which showed inhibition were then assayed
42
over a narrower concentration range to determine the IC50
.
Protein assays. Total protein estimations were performed using a
Coomassie Blue binding assay with BSA as standard (Pierce Chemical
Co., IL, USA).
Preparation of supernatants containing secreted phospholipase
activities. This was carried out essentially as described previously.40
Briefly, WT C. neoformans strain H99 was grown to confluence on SDA in
16 cm diameter Petri dishes for 72 h at 30 °C in air. Cells scraped from 10-
20 dishes were washed sequentially in 50 ml Falcon tubes with isotonic
saline and imidazole buffer (10 mM imidazole, 2 mM CaCl2, 2 mM MgCl2,
56 mM D-Glucose, made up in isotonic saline, pH 5.5). The final cell pellet
was resuspended in a volume of imidazole buffer equivalent to about 10 %
of the cell volume, and incubated for 24 h at 37 °C. The cell-free
supernatant was collected by centrifugation and stored at -70 °C.
Assessment of fungal mitochondrial function by DiOC6 staining. C.
neoformans cells were grown in YPD broth overnight and resuspended in
fresh YPD medium at OD600=0.1. Cultures were supplemented with
compounds 1 or 8 at 2-4 µg/ml or FCCP at 3 µM. For the flow cytometry,
the cultures were incubated for 3.5 hours and the OD of each culture was
adjusted to 0.06 using the same (used) medium. The cells were then
stained with 0.3 µM DiOC6 for 20 min and analyzed using BD FACSCanto
II. The assay was performed in triplicate. For microscopy, similarly treated
cultures were incubated for 7 hours and their OD adjusted to 0.5 using the
same (used) medium. The cells were then stained with DiOC6 (2.5 µM)
and DAPI (1 µg/ml) for 15 min. The cells were visualized using DeltaVision
RT deconvolution microscope (Applied Precision, Inc., Issaquah, WA)
fitted with a 100 oil/1.40 objective lens (Olympus, Tokyo, Japan).
Radiometric assay for fungal phospholipase activity. The three
enzyme activities associated with the multifunctional enzyme, PLB1 and
present in the cryptococcal supernatant (PLB: hydrolyses both fatty acyl
chains from a phospholipid; LPL: hydrolyses the remaining fatty acyl chain
on
a lysophospholipid and LPTA: transfers a fatty acid onto a
lysophospholipid to reconstitute a phospholipid) were measured as
described previously8,11,40 in a final volume of 125 µL at 37° C. For the
determination
of
secreted
PLB
activity, carrier
dipalmitoyl
phosphatidylcholine (DPPC, final concentration 800 µM) and 1,2-di[1-14C]
palmitoyl phosphatidylcholine (20,000 dpm) were dried under nitrogen and
suspended in 125 mM imidazole acetate buffer pH 4.0 (assay buffer) by
sonication using a Branson 450 sonifier. Each reaction contained 1 µg of
total protein and the reaction time was 22 min. PLB activity was determined
by the rate of decrease of the radiolabelled PC substrate, with appearance
of the label in free fatty acid. Secreted LPL and LPTA activities were
measured simultaneously in a reaction mixture containing 1-[14C]palmitoyl
lyso-PC (25,000 dpm) and carrier lyso-PC (final concentration 200 µM) in
assay buffer. Each reaction contained 1 µg of total protein and the reaction
time was 15 sec. LPL activity was measured by the rate of loss of 1-
Acknowledgements
This work was supported by the National Health and Medical
Research Council of Australia project grant number 402413. D.
Obando was supported by a post-graduate stipend from the
Centre for Infectious Diseases and Microbiology, The University
of Sydney, Westmead Hospital and the School of Chemistry, The
University of Sydney. TCS is
Foundation Fellow.
a Sydney Medical School
[
14C]palmitoyl lyso-PC, with appearance of the label in free fatty acid. LPTA
activity was estimated from the rate of formation of radiolabelled DPPC.
13
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10.1002/cmdc.201800331
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FULL PAPER
[27] Reference method for broth dilution susceptibility testing of filamentous
fungi: Approved Standard. NCCLS document M38-A (ISBN 1-56238-
186-5). National Committee for Clinical Laboratory Standards,
Pennsylvania, USA, 2002.
Keywords: medicinal chemistry • structure-activity
relationships • antifungal agent • bispyridinium • haemolytic
activity
[28] G. Malich, B. Markovic, C. Winder, Toxicology 1997, 124, 179-192.
[29] R. Ganendren, F. Widmer, V. Singhal, C. Wilson, T. C. Sorrell, L. Wright,
Antimicrob. Agents Chemother. 2004, 48, 1561-1569.
[30] G. H. De Haas, M. G. Van Oort, R. Dijkman, R. Verger, R. Biochem. Soc.
Trans. 1989, 17, 274-276.
References:
[31] M. Chayakulkeeree, S. A. Johnston, J. Bijosono Oei, S. Lev, P. R.
Williamson, C. F. Wilson, X. Zuo, A. L. Leal, M. H. Vainstein, W.
Meyer, T. C. Sorrell, R. C. May, J. T. Djordjevic, Mol. Microbiol. 2011,
80, 1088–1101.
[1]
a) A. Chakrabati, M. R. Shivaprakash, J. Postgrad. Med. 2005, 51, 16-
20; b) F. Zand, M. Moghaddami, M. A. Davarpanah, M. Masjedi, R.
Nikandish, A. Amanati, M. Afarid, L. Nafarieh, M. Nami, Biosci. Biotech.
Res. Comm. 2016, 9, 371-381; c) K. V. Ramana, S. Kandi, V.
Bharatkumar, C.H. V. Sharada, R. Rao, R. Mani, S. D. Rao, Am. J. Infect.
Dis. and Microbiol., 2013, 1, 64-69.
[32] F. Wang, M. A. Ogasawara, P. Huang, Mol. Aspects Med. 2010, 31, 75–
92.
[33] a) M. Shingu-Vazquez, A. Traven, Eukaryotic Cell, 2011, 10, 1376–1383;
b) R. Calderone, D. Li, A. Traven, FEMS Yeast Res., 2015, 15, fov027;
doi: 10.1093/femsyr/fov027
[2]
R. Rajasingham, R. M. Smith, B. J. Park, J. N. Jarvis, N. P. Govender, T.
M. Chiller, D. W. Denning, A. Loyse, D. R. Boulware, Lancet Infect. Dis.,
2017, 17, 873-881.
[34] a) X. Zuo, J. T. Djordjevic, J. Bijosono Oei, D. Desmarini, S. D. Schibeci,
K. A. Jolliffe, T. C. Sorrell, Mol. Pharmacol., 2011, 80, 476-485; b) J. H.
Kim, R. P. Haff, N. C. G. Faria, M. de L. Martins, K. L. Chan, B. C.
Campbell. Molecules, 2013, 18, 8873-8894; c) M. A. Ansari, Z. Fatima,
S. Hameed, Open Microbiol. J., 2016, 10, 12-22; d) J. H. Kim, H.-O. Lee,
Y.-J. Cho, J. Kim, J. Chun, J. Choi, Y. Lee, W. H. Jung, PLOS ONE, 2014,
9(2): e89122.
[3]
[4]
E. L. Berkow, S. R. Lockhart, Infect. Drug Resist., 2017, 10, 237-245.
a) M. Nucci, E. Anaissie, Clin. Microbiol. Rev., 2007, 20, 695-704;b) D.
L. Horn, A. G. Freifeld, M. G. Schuster, N. E. Azie, B. Franks, C. A.
Kauffman. Mycoses, 2014, 57, 652–658. c) A. Pellon, A. Ramirez-Garcia
I. Buldain, A. Antoran, A. Rementeria, F. L. Hernando, PloSOne, 2017,
12, e0174885.
[5]
[6]
J. T. Djordjevic, Frontiers in Microbiology, 2010, 1, 1-13.
F. Mirbod, Y. Banno, M. A. Ghannoum, A. S. Ibrahim, S. Nagashima, Y.
Kitajhima, G. T. Gole, Y. Nozawa, Biochim. Biophys. Acta 1995, 1257,
181-188.
[35] H. Chang, H. Huang, T. Huang, P. Yang, Y. Wang, H. Juan, Bio-protocol,
2013, 3 e430.
[36] P. Stöckl, C. Zankl, E. Hütter, H. Unterluggauer, P. Laun, G. Heeren, E.
Bogengruber, D. Herndler-Brandstetter, M. Breitenbach, P. Jansen-Dürr,
Free Radic. Biol. Med., 2007, 43, 947-958.
[7]
[8]
R. A. Calderone, W. A.; Fonzi, Trends Microbiol. 2001, 9, 327-335.
S. C. A. Chen, M. Muller, J. Z. Zhou, L. C. Wright, T. C.; Sorrell, T. C. J.
Infect. Dis. 1997, 175, 414-420.
[37] M. Korchak, A. M. Rich, C. Wilkenfield, L. E. Rutherford, G. Weissmann,
Biochem. Biophys. Res. Comm., 1982, 108, 1495-1501.
[38] M. Kimara, I. Kimura, M. Muroi, K. Tanaka, H. Nojima, T. Uwano, T.
Koizumi Biol. Pharm. Bull., 1994, 17, 1224-1231.
[9]
G. M. Cox, H. C. McDade, S. C. A. Chen, S. C. Tucker, M. Gottfredsson,
L. C. Wright, T. C. Sorrell, S. D. Leidich, A. Casadevall, M. A. Ghannoum,
J. R. Perfect, J. R. Mol. Microbiol. 2001, 39, 166-175.
[39] R. Hulst, I. Muizebelt, P. Oosting, C. Van der Pol, A. Wagenaar, J.
Smisterova, E. Bulten, C. Driessen, D. Hoekstra, J. B. F. N. Engberts,
Eur. J. Org. Chem., 2004, 4, 835-849.
[10] D. K. Shen A. D. Noodeh A. Kazemi, R. Grillot, G. Robson, J. F. Brugère,
FEMS Microbiol. Lett., 2004, 239, 87-93.
[11] S. C. Chen, L. C. Wright, J. C., Golding, T. C. Sorrell, Biochem. J., 2000,
347, 431–439.
[40] S. C. Chen, L. C. Wright, R. T. Santangelo, M. Muller, V. R. Moran, P. W.
Kuchel, T. C. Sorrell, Infect. Immun. 1997, 65, 405-411.
[12] C. K. L. Ng, D. Obando, F. Widmer, L. Wright, T. C. Sorrell and K. A.
Jolliffe, J. Med. Chem., 2006, 49, 811-816.
[41] E. C. Bligh, W. J. Dyer, Can. J. Biochem. Physiol. 1959, 37, 911-917.
[42] GraphPad Prism version 4.02 for Windows, GraphPad Software, San
Diego California, USA, www.graphpad.com
[13] C. K. L. Ng, V. Singhal, F. Widmer, L. C. Wright, T. C. Sorrell and K. A.
Jolliffe, Bioorg. Med. Chem., 2007, 15, 3422-3429.
[14] D. Obando, N. Pantarat, R. Handke, Y. Koda, F. Widmer, J. Djordjevic,
D. H. Ellis, T. C. Sorrell and K. A. Jolliffe, Bioorg. Med. Chem., 2009, 17,
6329-6339.
[15] S. C.-A. Chen, C. Biswas, R. Bartley, F. Widmer, N. Pantarat, D. Obando,
J. T. Djordjevic, D. H. Ellis, T. C. Sorrell, K. A. Jolliffe, Antimicrob. Agents
Chemother., 2010, 54, 3233-3240.
[
[16] S. Gunzenhauser, E. Biala, P. Strazewski, Tetrahedron Lett., 1998, 39,
6277-6280.
[17] J. F. W. Keana, G. S. Heo, J. S. Mann, F. L. Van Nice, L. Lex, V. S.
Prabhu, G. Ferguson, J. Org. Chem., 1988, 53, 2268-2274.
[18] E. C. Hass, P. G. Mezey, S. R. Abrams, J. Comput. Chem., 1982, 3, 185-
190.
[19] D. Ma, X. Lu, Tetrahedron, 1990, 46, 6319-6330.
[20] S. W. Baldwin, J. D. Wilson, J. Aube, J. Org. Chem., 1985, 50, 4432-
4439.
[21] L. Brandsma, W. F. Nieuwenhuizen, J. W. Zwikker, U. Mäeorg. Eur. J.
Org, Chem., 1999, 5, 775-779.
[22] J. M. Montierth, D. R. DeMario, M. J. Kurth, N. E. Schore, Tetrahedron,
1998, 54, 11741-11748.
[23] A. Lei, M. Srivastava, X. Zhang, J. Org. Chem., 2002, 67, 1969-1971.
[24] R. Chinchilla, C. Nájera, Chem. Soc. Rev., 2011, 40, 5084-5121.
[25] T. Ueda, N. Kanomata, H. Machida, Org. Lett., 2005, 7, 2365-2368.
[26] Reference method for broth dilution susceptibility testing of yeasts:
Approved Standard. NCCLS document M27-A (ISBN 1-56238-186-5).
National Committee for Clinical Laboratory Standards, Pennsylvania,
USA, 1997.
14
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Entry for the Table of Contents
A series of bis(4-pentylpyridinium) compounds with a variety of linkers between the two head groups have been synthesised and their
antifungal activity evaluated, together with their haemolytic activity and mammalian cell cytotoxicity. An ortho-dihexylbenzene spacer
provided good antifungal activity and an improved therapeutic index over the dodecanyl and tetradecanyl derivatives.
15
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