Synthesis and biocompatibility evaluation of partially fluorinated pyridinium bromides

Article abstract of DOI:10.1039/b516039a

Although cationic surfactants are of general interest for a variety of consumer and biomedical applications, only a limited number of partially fluorinated, single-tailed, cationic surfactants have been synthesized. To study the potential usefulness of fluorinated cationic surfactants for these applications we synthesized a series of partially fluorinated pyridinium bromide surfactants. Three 10-perfluoroalkyldecyl pyridinium surfactants were synthesized by coupling a perfluoroalkyl iodide with 9-decene-1-yl acetate using an AIBN mediated radical reaction. The resulting 9-iodo-10-perfluoroalkyldec-1- yl acetates were deiodinated using HI-Zn-EtOH and hydrolyzed using KOH-EtOH to yield the corresponding 10-perfluoroalkyldecanol. The partially fluorinated alcohol was converted into the bromide using Br₂-PPh₃. Alkylation of excess pyridine with the bromides gave the desired 10-perfluoroalkyldecyl pyridinium bromides in good yields. Three 10-perfluoroalkylundecyl surfactants were synthesized using a similar approach with 10-undecenoic acid methyl ester as starting material. Based on an initial in vitro toxicity assessment, the toxicity of the partially fluorinated pyridinium surfactants was slightly lower or comparable to benzalkonium chloride, a typically cationic surfactant (with IC₅₀s of tested compounds ranging from 5 to 15 μM). An increase in the length and/or the degree of fluorination of the hydrophobic tail correlated with a mild decrease of cytotoxicity and haemolytic activity. Partially fluorinated pyridinium surfactants may, therefore, be useful for biomedical applications such as components for novel gene and drug delivery systems. the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006.

Full text of DOI:10.1039/b516039a

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PAPER
www.rsc.org/njc | New Journal of Chemistry
Synthesis and biocompatibility evaluation of partially fluorinated
pyridinium bromides
a
b
tigova, Andrea Kasna
¨ ´ ´
ˇ
,b
b
Sandhya M. Vyas, Jaroslav Tura
´
b
nek, Pavlı
´
c
na Kno
, Venkat Koganti, Stephen E. Rankin, Barbara L. Knutson
c
c
Veronika Kvardova
´
and Hans-Joachim Lehmler*
a
Received (in St Louis, MO, USA) 11th November 2005, Accepted 3rd April 2006
First published as an Advance Article on the web 26th April 2006
DOI: 10.1039/b516039a
Although cationic surfactants are of general interest for a variety of consumer and biomedical
applications, only a limited number of partially fluorinated, single-tailed, cationic surfactants have
been synthesized. To study the potential usefulness of fluorinated cationic surfactants for these
applications we synthesized a series of partially fluorinated pyridinium bromide surfactants. Three
1
0-perfluoroalkyldecyl pyridinium surfactants were synthesized by coupling a perfluoroalkyl
iodide with 9-decene-1-yl acetate using an AIBN mediated radical reaction. The resulting 9-iodo-
0-perfluoroalkyldec-1-yl acetates were deiodinated using HI–Zn–EtOH and hydrolyzed using
1
KOH–EtOH to yield the corresponding 10-perfluoroalkyldecanol. The partially fluorinated
alcohol was converted into the bromide using Br –PPh . Alkylation of excess pyridine with the
2
3
bromides gave the desired 10-perfluoroalkyldecyl pyridinium bromides in good yields. Three 10-
perfluoroalkylundecyl surfactants were synthesized using a similar approach with 10-undecenoic
acid methyl ester as starting material. Based on an initial in vitro toxicity assessment, the toxicity
of the partially fluorinated pyridinium surfactants was slightly lower or comparable to
benzalkonium chloride, a typically cationic surfactant (with IC50s of tested compounds ranging
from 5 to 15 mM). An increase in the length and/or the degree of fluorination of the hydrophobic
tail correlated with a mild decrease of cytotoxicity and haemolytic activity. Partially fluorinated
pyridinium surfactants may, therefore, be useful for biomedical applications such as components
for novel gene and drug delivery systems.
Introduction
Although hydrocarbon cationic surfactants such as benzalk-
onium chloride (BAC) and hexadecylpyridinium chloride are
of considerable interest for a broad range of consumer and
biomedical applications (for example, antimicrobial agents in
Fluorinated surfactants are highly surface active, display weak
intermolecular interactions, and typically have only low acute
toxicity, properties which make them highly suitable for a
large number of technical and, potentially, biomedical appli-
17,18
18
mouthwashes,
ophthalmic and contact lens solutions
19–21
and novel catanionic gene or drug delivery systems
) only
1
cations. Because of these unique properties there is, for
a small number of fluorinated cationic surfactants have been
synthesized and their biocompatibility assessed. More work is,
therefore, needed to determine how the biocompatibility of a
partially fluorinated, cationic surfactant is different from its
hydrocarbon analogue and how structural features, for exam-
ple the chain-length and the degree of fluorination of the
hydrophobic tail, influence a surfactant’s biocompatibility.
The aim of this work was to develop an efficient synthetic
route to partially fluorinated pyridinium surfactants and to
perform an initial toxicity assessment to test their potential
usefulness for novel drug delivery systems and other biomedi-
cal applications.
example, considerable interest in fluorinated cationic surfac-
tants as templates for the synthesis of ordered porous cera-
2
–7
mics.
Other ongoing research is focusing on the use of
,9
8
fluorinated surfactants in biomedical applications. Single
tailed partially fluorinated surfactants with a variety of non-
1
0
ionic or zwitterionic head groups, for example carnitine,
1
morpholinophosphate,
1
and carbohydrate
1
12
2–7
phosphocholine,
pyridinium,
3–16
head groups, have been synthesized.
In contrast to their hydrocarbon counterparts, many partially
fluorinated surfactants, especially surfactants with a high
degree of fluorination, display low or moderate toxicity in
in vitro and in vivo studies and are, thus, considered to be
biocompatible.
Results and discussion
a
Department of Occupational and Environmental Health, University
of Iowa, Iowa City, IA 52242, USA. E-mail: hans-joachim-
Synthesis
lehmler@uiowa.edu; Fax: (þ1) 3193354290; Tel: (þ1) 3193354211
Most partially fluorinated compounds are not available from
commercial sources. One crucial step in the synthesis of any
partially fluorinated surfactants is, therefore, the synthesis of a
functionalized partially fluorinated alkyl chain. Such partially
b
Veterinary Research Institute, Department of Immunology, Hudcova
70, 621 33 Brno, Czech Republic
c
Chemicals and Materials Engineering Department, University of
Kentucky, Lexington, KY 40536, USA
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the alcohols 9a–c from approximately 60% to 70%. In the case
of the acetate series, the alcohols 10a–c were obtained by
hydrolysis of the acetate followed by recrystallization from
2
3
hexane. Typical overall yields of the alcohols 10a–c were
6
5–85%.
The alcohols 9a–c and 10a–c were brominated using triphe-
nylphosphine and bromine similar to the procedure reported
2
4
by Naud and co-workers. Bromination of the alcohols 9a–c
and 10a–c with HBr/H SO also yielded the desired partially
2
4
fluorinated bromides, but this approach was abandoned be-
cause the purification of the bromides is tedious and the yields
are low. In the final step of the synthesis excess pyridine and
the respective bromides were heated under reflux to yield the
Scheme 1 a. CH
55%), Zn, C OH, D; d. (i) CH
ether, ambient temperature; e. LiAlH
temperature; f. PPh , Br , DCM, ambient temperature; g. pyridine, D.
3
OH, PTSA, toluene, D; b. R
OH, KOH, D; (ii) LiAlH
, anhydrous ether, ambient
F
I, AIBN, D; c. HI
(
H
2 5
3
4
, anhydrous
4
3
2
2
6
corresponding pyridinium bromides 13a–c and 14a–c.
1
The H NMR spectra of most of the pyridinium bromides
showed one singlet around d ¼ 3.1 ppm which was not related
to any group in the compound. The chemical shift of this peak
was not consistent, thus suggesting that, like their hydrocar-
bon analogues, several of the partially fluorinated pyridinium
fluorinated alkyl moieties are typically synthesized by the
coupling of a perfluorinated iodide with an o-unsaturated
hydrocarbon precursor in an AIBN mediated radical reac-
2
2–25
tion.
We have utilized this approach to synthesize a series
1
of terminally fluorinated pyridinium bromides.
salts form stable monohydrates. This interpretation of the H
As shown in Schemes 1 and 2, the iodo methyl esters 5a–c or
iodoacetates 6a–c were synthesized using this reaction from
undecenoic methyl ester 3 or decenol acetate 4, respectively.
Addition of AIBN in multiple portions was a key factor in this
reaction. The secondary iodides 5a–c or 6a–c were deiodinated
using HI–Zn–EtOH without further purification. Hydroiodic
acid and zinc were chosen because both reagents are easy to
NMR spectra was further confirmed by D O exchange experi-
2
ments. In addition, the results from the combustion analysis
experiments confirm the presence of one water molecule in the
pyridinium salts 13a and 14a–c.
Cytotoxicity and haemolytic assessments
2
4
handle and less toxic compared to tributyltin hydride, which
is typically employed in similar deiodination reactions.
As shown in Scheme 1, the series of methyl esters 7a–c was
converted into the corresponding alcohols 9a–c by two differ-
ent routes. Initially the esters 7a–c were converted into the
Despite their importance for a variety of consumer and
biomedical applications, only limited information about the
toxicity of cationic surfactants, especially fluorinated cationic
1
8
surfactants, is available. An initial cytotoxicity assessment of
all six pyridinium surfactants was therefore performed using
2
7
corresponding, R
F
-substituted (i.e. partially fluorinated) un-
the A2780 ovarian carcinoma cell line. The IC50 values of the
pyridinium salts and several other surfactants are summarized
in Tables 1 and 2, respectively. The partially fluorinated
pyridinium surfactants (13a–c and 14a–c) were highly toxic
in the A2780 cancer cell line with IC50 values ranging from 5 to
15 mM. These IC50 values are comparable to IC50 values of
2
2
decanoic acids which were subsequently reduced to the
alcohols 9a–c with lithium aluminium hydride in anhydrous
ether (reaction condition d in Scheme 1). This approach was
chosen because the partially fluorinated acids can be more
easily purified by recrystallization from hexanes compared to
2
2,23
27
structurally related partially fluorinated alcohols,
thus
typical anticancer agents, e.g. platinum complexes. The most
allowing one to perform the synthesis on a large (100 gram)
scale. To further improve the overall yield of the alcohols we
optimized the direct reduction of the methyl ester 7a–c with
lithium aluminium hydride (reaction condition e in Scheme 1).
The direct reduction of the esters 7a–c increased the yield of
toxic compounds 13a and 14a were also bactericidal against
Salmonella typhimurium (results not shown). Overall, the
low IC50 values of 13a–c and 14a–c are in agreement with
a
previous report that highly fluorinated 1H,1H,2H,
2H,3H,3H-perfluoroundecyltrialkylammonium salts are toxic
2
in Namalva lymphoblastoid cells with IC50s below 0.16 mM.
8
The short-chain pyridinium salts 13a and 14a were the most
toxic surfactants in this series with toxicities comparable with
BAC. Similar results were obtained with several other cell lines
(
data not shown). The increase in the length of the partially
fluorinated tail caused a mild decrease in cytotoxicity, thus
suggesting that the toxicity of cationic surfactants can be
reduced by introducing a certain degree of fluorination. This
hypothesis is further supported by the observation that the
partially fluorinated pyridinium surfactant 14a was less toxic
compared to its hydrocarbon analogue tetradecylpyridinium
bromide. This hydrocarbon surfactant was the most toxic
compound investigated in our study. 1H,1H,2H,2H-perfluoro-
dodecylpyridinium chloride, a highly fluorinated pyridinium
surfactant with a slightly shorter hydrophobic tail compared
Scheme 2 a. DMAP, AcCl, pyridine, DCM; b. R
55%), Zn, C OH, D; d. CH OH, KOH, D; e. PPh
ambient temperature; f. pyridine, D.
F
I, AIBN, D; c. HI
(
2
H
5
3
, Br , DCM,
3 2
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New J. Chem., 2006, 30, 944–951 | 945

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Table 1 Haemolytic and cytotoxicity assessment of the partially fluorinated pyridinium bromides 14a–c and 15a–c and related cationic
fluorocarbon and hydrocarbon surfactants in the A2780 cell line
Compound
A2780 cell line IC50 [mM]
.97
Haemolysis PBS [mM]
Haemolysis PBS þ 20% FBS [mM]
7
62.5
250
1
1
1.1
4.7
62.5
62.5
62.5
62.5
62.5
250
250
125
4
9
.9
.3
250
125
1
3
1.7
1.4
125
4500
2
5
.3
.3
62.5
125
250
400
to 14a, was the least toxic cationic surfactant, an observation
that also suggests that an increased degree of fluorination may
decrease the cytotoxicity of pyridinium surfactants.
and PBS containing 20% fetal bovine serum. As with the
cytotoxicity, it is well established that the in vitro haemolytic
activity of fluorinated surfactants on red blood cells dramati-
cally decreases with increasing degree of fluorination and
1
8
In agreement with previous studies, the partially fluori-
nated cationic surfactants 13a–c and 14a–c (Table 1) were
much more toxic compared to typical anionic surfactants and
several non-ionic surfactants with a hydrocarbon tail (Table
1
1–14,29–31
increasing length of the fluorinated hydrophobic tail.
In contrast, the haemolytic activity of hydrocarbon surfac-
tants typically increases with increasing chain length. The
pyridinium surfactants showed significantly stronger haemo-
lytic activity than BAC (Table 1) and the anionic and non-
ionic surfactants (Table 2) investigated. This observation is in
agreement with the strong haemolytic activity reported for
2
). Some non-ionic surfactants, e.g. Triton X-100 or Nonidet
P40, were also toxic towards A2780 cells in a range compar-
able to that of the partially fluorinated pyridinium surfactants.
The mechanisms of the relatively high toxicity of cationic
surfactants in general and of the partially fluorinated surfac-
tants 13a–c and 14a–c in particular most likely involve a
disruption of the integrity of the cell membrane. This hypoth-
esis is supported by the fact that the partially fluorinated
surfactants are equally toxic in several cell lines and in
bacteria, e.g. Salmonella typhimurium (data not shown).
Because of the potential biomedical use of the partially
fluorinated surfactants 13a–c and 14a–c we also investigated
their haemolytic activity on rabbit red blood cells in both PBS
3
5
highly fluorinated trialkylammonium salts. The lowest con-
centration of the pyridinium surfactants at which haemolytic
activity was observed did not appear to depend on the degree
of fluorination and/or the length of the hydrophobic tail. One
exception was the highly fluorinated 1H,1H,2H,2H-perfluoro-
dodecylpyridinium chloride which exhibited haemolytic activ-
ity at concentrations comparable to several anionic and non-
ionic surfactants, thus suggesting that, in contrast to the
cytotoxicity studies, a significant degree of fluorination may
9
46 | New J. Chem., 2006, 30, 944–951 This journal is ꢀc the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006

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Table 2 Haemolytic and cytotoxicity assessment of typical anionic and non-ionic surfactants in the A2780 cell line
Compound
A2780 cell line IC50 [mM]
Haemolysis PBS [mM]
Haemolysis PBS þ 20% FBS [mM]
41000
2
94
250
2
43
41000
41000
5
6
300
500
4500
4500
6
80
6
2
8
125
830
41000
41000
2.3
2
60
41000
41000
1
1.2
400
830
be necessary before a protective effect against haemolysis can
be observed.
therefore, be useful for biomedical applications such as com-
ponents for novel gene and drug delivery systems.
Conclusion
Experimental
These findings suggest that partial fluorination of the hydro-
phobic tail of pyridinium and other cationic surfactants may
reduce their toxicity and haemolytic activity. This observation
is comparable to single tailed non-ionic and zwitterionic
surfactants, where partial fluorination of the hydrophobic tail
results in a reduction of the toxicity compared to the hydro-
carbon analogue. Despite their cytotoxicity, cationic surfac-
tant-lipids are used for the construction of synthetic delivery
systems, e.g. liposomes, for transfection of cells by DNA/
Perfluorinated iodides were purchased from Oakwood Che-
mical Co. (West Columbia, South Carolina, USA) and used as
received. Long-chain hydrocarbon starting materials were
purchased from TCI Chemicals (Portland, Oregon, USA).
Anhydrous solvents were purchased from Fisher Scientific
1
(Fairlawn, New Jersey, USA). All the H NMR spectra were
3
recorded in CDCl on a multinuclear Bruker Avance 300
Digital NMR spectrometer at 297 K; all chemical shifts are
reported in parts per million (ppm) relative to internal tetra-
3
2
RNA constructs. Fluorinated, cationic surfactants may,
methylsilane (Me Si), with the residual solvent proton
4
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New J. Chem., 2006, 30, 944–951 | 947

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resonance as internal standard. Coupling constants are given
hours under stirring. After complete conversion it was cooled
to 0 1C and water was added dropwise. The mixture was
acidified with 2N H SO and extracted with diethyl ether (3 ꢂ
1
in hertz (Hz). F chemical shifts were determined using CFCl
9
3
as internal standard. Melting points were determined using a
Thermal Analysis 2920 Differential Scanning Instrument.
2
4
100 ml). The solution was washed with water (2 ꢂ 100 ml),
ꢁ
1
Thermograms were recorded using a heating rate of 201 min
from 20 to 230 1C and the temperature maxima of the main
saturated aqueous NaHCO solution (1 ꢂ 100 ml), and brine
3
(1 ꢂ 100 ml), and then dried over MgSO . Removal of the
4
2
2
phase transitions after one DSC run were determined. Mass
spectra on non-ionic intermediates were measured at the
University of Iowa Mass Spectrometry Facility using a Voya-
ger GC-MS instrument operated at 70 eV in the electron
impact mode. The reported fragment peaks correspond to
the most abundant ions, in addition to the parent ion(s). Mass
spectra of pyridinium salts were recorded at the University of
Kentucky Mass Spectrometry Facility using a Bruker Autoflex
time-of-flight mass spectrometer equipped with MALDI. Mi-
croanalytical data were obtained from Atlantic Micro Lab
Microanalysis Service (Atlanta, Georgia, USA). IR spectra
were measured with a ThermoNicolet Nexus 470 FT-IR
spectrophotometer.
solvent by rotary evaporation afforded the crude acids or
alcohols which were purified by recrystallization with hex-
2
2,23
ane.
The analytical data of the acids and alcohol 10b are in
3
agreement with previously published data.
2
1
1
1,11,12,12,13,13,14,14,14-Nonafluorotetradecanol (10a). H
NMR d/ppm 1.34 (m, 12H, –(CH –), 1.57 (m, 4H,
CH CH CH OH), 1.9–2.4 (m, 2H þ OH,
– and ꢁCH
R CH CH –), 3.66 (t, J ¼ 6.6 Hz, 2H, ꢁCH CH OH).
2 6
)
R
F
2
2
2
2
1
3
C
F
2
2
2
2
NMR d/ppm 20.1 (t, J ¼ 4.0 Hz), 25.7, 29.1, 29.2, 29.3, 29.4,
1
9
2
9.5, 30.8 (t, J ¼ 22.0 Hz), 32.7, 62.9. F NMR d/ppm ꢁ81.6
CF ), ꢁ115.1, ꢁ124.9, ꢁ126.6. GC/MS m/z (relative inten-
(
3
sity, %) 358 (M ꢁ H
2
0, 5), 330 (30), 302 (20), 288 (30).
ꢁ
1
IR(KBr) n/cm 3346 (OH), 2930, 2858, 1235, 1133, 1117, 722.
AIBN mediated coupling reaction (general procedure)
1
1,11,12,12,13,13,14,14,15,15,16,16,17,17,18,18,18-Hepta-
1
3
The radical addition of R I to undecenoic methyl ester 3 or
3
F
decafluorooctadecanol (10c). H NMR d/ppm 1.35 (m, 12H,
(CH
3
4
decenol acetate 4 in the presence of AIBN was performed
–
1
6
2
)
6
–), 1.59 (m, 4H, R
F
CH
2
CH
2
– and ꢁCH
CH CH
–), 3.68 (t, J ¼
OH). C NMR d/ppm 20.0 (t, J ¼ 3.8
2 2
CH OH),
2
3,35,36
following literature procedures.
methyl ester 3 (98 g, 0.5 mol) or alkenol acetate 4 (91 g, 0.5
mol) and a perfluorinated iodide R I (R , C
¼ C
17, 0.55 mol) were placed in a 500 ml round bottomed flask
equipped with a reflux condenser. The radical initiator AIBN
2 mol%) was added. The reaction mixture was heated to 90
C under stirring. After 30 min, the flask was cooled to room
The alkenoic acid
.88 (br s, 1H, OH), 2.08 (m, 2H, R
F
2
2
13
.7 Hz, 2H, ꢁCH
2 2
CH
F
F
4
F
9
6 13
F ,
Hz), 25.7, 29.1, 29.2, 29.3, 29.4, 29.5, 30.8 (t, J ¼ 22.5 Hz),
C
8
F
19
2.7, 62.9. F NMR d/ppm ꢁ81.5 (CF ), ꢁ115.1, ꢁ122.3 (3 ꢂ
3
3
CF ), ꢁ123.3, ꢁ124.1, ꢁ126.7. GC/MS m/z (relative intensity,
2
(
%
) 558 (M ꢁ H O, 2) 530 (10), 502 (5), 488 (9). IR(KBr)
2
1
ꢁ1
n/cm 3322 (OH), 2933, 2855, 1242, 1205, 1150, 1117, 661.
temperature, and another portion of AIBN (2 mol%) was
added. The stirred solution was again heated to 90 1C, and the
entire process was repeated at least two additional times; 3 h
were allowed to pass after the final addition of AIBN. The
Reduction of partially fluorinated acids and esters with LiAlH4
(general procedure)
The partially fluorinated acids or esters (0.15 mol) were
dissolved in 50–80 ml of THF and added over 5 min to a
1
progress of the reaction was monitored by GC and H NMR
spectroscopy of small aliquots collected between AIBN addi-
tions. The flask was cooled to room temperature, and the
crude iodo methyl esters 5a–c or iodoacetates 6a–c were used
in the next step without further purification.
slurry of LiAlH (3.8 g, 0.1 mol) in 10 ml THF at 0 1C under
4
nitrogen. The mixture was allowed to come to room tempera-
ture and was stirred overnight. After complete conversion it
was cooled to 0 1C and water was added dropwise until the
evolution of hydrogen ceased. The mixture was acidified with
Terminally perfluorinated esters 7a–c and acetates 8a–c
2
N HCl and extracted with diethyl ether (3 ꢂ 100 ml). The
solution was washed with water (2 ꢂ 100 ml), saturated
aqueous NaHCO
solution (1 ꢂ 100 ml), and brine (1 ꢂ 100
ml), and then dried over MgSO . Removal of the solvent by
(
general procedure)
The crude secondary iodides 5a–c or 6a–c were dissolved in
3
1
2 5
50 ml of C H OH and hydroiodic acid (non-stabilized, 55%,
4
3
5 ml, 1.5 mol) was added. Zinc dust (97 g, 1.5 mol) was added
rotary evaporation afforded the crude alcohols (9a–c), which
were purified by chromatography on silica gel (hexane–ethyl
acetate ¼ 9 : 1) or recrystallization with hexane. Typical yields
ranged from 80–85%. The analytical data of 9a–c are in
very slowly at 0 1C. The mixture was refluxed for 6 h and then
filtered. The filtrate was evaporated to dryness and re-dis-
solved in diethyl ether (ca. 200 ml). The solution was washed
2
4
with water (2 ꢂ 100 ml), saturated aqueous NaHCO solution
3
agreement with previously published data.
(
1 ꢂ 100 ml), and brine (1 ꢂ 100 ml), and then dried over
Terminally perfluorinated bromides 11a–c and 12a–c
general procedure)
MgSO . Removal of the solvent afforded the crude terminally
4
(
fluorinated ester 7a–c or acetate 8a–c. The complete conver-
1
sion was confirmed by H NMR spectroscopy and GC.
Triphenylphosphine (24.2 g, 0.13 mol) and bromine (7 ml, 0.13
mol) were dissolved in 50 ml of anhydrous methylene chloride
at 0 1C. After 30 min, a DCM solution of the alcohol (9a–c or
10a–c, 0.1 mol in 25 ml of DCM) was added, and the solution
was stirred at room temperature for 15 h. The solution was
poured into ice water (300 ml). The product was extracted with
Hydrolysis of 7a–c and 8a–c (general procedure)
The partially fluorinated esters 7a–c or acetates 8a–c were
dissolved in 150 ml of C H OH. To this KOH (57 g, 1.5 mol)
2
5
was added slowly. The reaction mixture was refluxed for six
9
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ꢁ
1
diethyl ether (4 ꢂ 100 ml). The solution was washed with water
ꢁ125.0, ꢁ126.6. MS m/z (calcd) 452 (452). IR(KBr) n/cm
2928, 2856, 1233, 1133. Anal. calcd for C20 O: C
NBr ꢃ H
43.63; H 5.27; N 2.54. Found: C 43.45; H 5.27; N 2.50%.
(
2 ꢂ 100 ml), saturated aqueous NaHCO
3
solution (1 ꢂ 100
H
27
F
9
2
ml), and brine (1 ꢂ 100 ml), and then dried over MgSO .
4
Removal of the solvent by rotary evaporation afforded the
crude bromide (11a–c, 12a–c), which was purified by chroma-
tography on silica gel (hexane–ethyl acetate ¼ 9 : 1) or recrys-
1
2,12,13,13,14,14,15,15,16,16,17,17,17-Pentadecafluorohep-
1
tadecylpyridinium bromide (13b). Mp 61.0 1C, 75.6 1C.
H
2
2,23
NMR d/ppm 1.2–1.6 (m, 14H, –(CH ) –), 1.57 (m, 2H,
tallization with hexane.
Yields were typically 70–75%. The
2 7
R CH CH –), 1.9–2.1 (m, 4H, R CH CH – and
2
analytical data of 11a–c are in agreement with previously
2
published data.
F
2
2
F
2
4
ꢁCH CH –py), 5.05 (t, J ¼ 7.2 Hz, 2H, ꢁCH CH –py),
2
2
2
2
8
.24 (t, J ¼ 6.6 Hz, 2H, H-3,5), 8.62 (t, J ¼ 7.8 Hz, 1H, H-
13
1
1,11,12,12,13,13,14,14,14-Nonafluorotetradecyl
1
bromide
4), 9.70 (d, J ¼ 5.7 Hz, 2H, H-2,6). C NMR d/ppm 19.89 (t, J
¼ 3.6 Hz), 25.89, 28.88, 28.90, 28.99, 29.11, 29.13, 29.20, 30.66
(
12a). H NMR d/ppm 1.24 (m, 12H, –(CH ) –), 1.53 (m,
2
6
1
9
2
H, R
CH
NMR d/ppm 20.2 (t, J ¼ 4.0 Hz), 28.3, 28.9, 29.2, 29.3, 29.4,
F
CH
2
CH
2
–), 1.79 (m, 2H, ꢁCH
2
CH
2
Br), 1.98 (m, 2H,
(t, J ¼ 21.8 Hz), 31.94, 61.77, 128.41, 145.14 (2 ꢂ C).
F
1
3
R
F
2
CH
2
–), 3.34 (t, J ¼ 6.9 Hz, 2H, ꢁCH
2 2
CH Br).
C
NMR d/ppm ꢁ81.7 (CF ), ꢁ115.2, ꢁ122.8, ꢁ123.6, ꢁ124.4,
ꢁ1
3
ꢁ126.9. MS m/z (calcd) 552 (552). IR(KBr) n/cm 2925,
1
9
2
9.5, 30.9 (t, J ¼ 22.0 Hz), 33.0, 33.9. F NMR d/ppm ꢁ81.6
CF
), ꢁ115.1, ꢁ125.1, ꢁ126.6. GC/MS m/z (relative inten-
sity, %) 438 (M ꢁ 1, o 1), 317 (23), 303 (30), 151 (12), 137
2855, 1239, 1205, 1144, 1123. Anal. calcd for C H F NBr:
2
2
27 13
(
3
C 41.77; H 4.27; N 2.21. Found: C 41.64; H 4.29; N 2.07%.
ꢁ1
12,12,13,13,14,14,15,15,16,16,17,17,18,18,19,19,19-Hepta-
1
decafluorononadecylpyridinium bromide (13c). Mp 78.1 1C. H
(
100), 135 (96). IR(KBr) n/cm 2930, 2854, 1283, 1236, 1134.
1
1,11,12,12,13,13,14,14,15,15,16,16,16-Heptadecafluorohex-
NMR d/ppm 1.2–1.6 (m, 14H, –(CH ) –), 1.62 (m, 2H,
R CH CH –), 1.9–2.1 (m, 4H, R CH CH – and
F 2 2 F 2 2
2
7
1
adecyl bromide (12b). H NMR d/ppm 1.3 (m, 12H, –(CH
.56 (m, 2H, R CH CH CH Br), 2.02
–), 1.83 (m, 2H, ꢁCH
m, 2H, R CH CH CH Br).
–), 3.38 (t, J ¼ 6.8 Hz, 2H, ꢁCH
C NMR d/ppm 20.7 (t, J ¼ 4.0 Hz), 28.1, 28.7, 29.0, 29.2,
2
)
6
–),
1
F
2
2
2
2
ꢁCH CH –py), 5.07 (t, J ¼ 7.2 Hz, 2H, ꢁCH CH –py),
2
2
2
2
(
1
F
2
2
2
2
8.22 (t, J ¼ 6.6 Hz, 2H, H-3,5), 8.59 (t, J ¼ 7.8 Hz, 1H, H-
3
13
4), 9.63 (d, J ¼ 5.4 Hz, 2H, H-2,6). C NMR d/ppm 20.20 (t,
1
9
2
9.3, 29.3, 30.9 (t, J ¼ 22.2 Hz), 32.8, 34.0. F NMR d/ppm
81.3 (CF ), ꢁ114.9, ꢁ122.5, ꢁ123.4, ꢁ124.1, ꢁ126.7.
J ¼ 3.6 Hz), 26.19, 29.19, 29.31, 29.42 (2 ꢂ C), 29.50 (2 ꢂ C),
1
9
ꢁ
30.68 (t, J ¼ 22.4 Hz), 32.21, 62.19, 127.63, 145.30, 145.36.
F
3
GC/MS m/z (relative intensity, %) 538 (M ꢁ 1, o1), 417
NMR d/ppm ꢁ81.2 (CF ), ꢁ114.8, ꢁ122.3 (3 ꢂ CF ), ꢁ123.1,
3
2
ꢁ
1
ꢁ1
(
14), 403 (20), 151 (12), 137 (100), 135 (95). IR(KBr) n/cm
932, 2858, 1241, 1207, 1145.
ꢁ124.0, ꢁ126.6. MS m/z (calcd) 652 (652). IR(KBr) n/cm
2922, 2854, 1245, 1204, 1147, 1115. Anal. calcd for
17NBr: C 39.36; H 3.68; N 1.91. Found: C 39.39; H
2
24 27
C H F
1
1,11,12,12,13,13,14,14,15,15,16,16,17,17,18,18,18-Hepta-
1
3
.66; N 1.80%.
decafluorooctadecyl bromide (12c). H NMR d/ppm 1.32 (m,
1
2H, –(CH ) –), 1.60 (m, 2H, R CH CH –), 1.86 (m, 2H,
2
11,11,12,12,13,13,14,14,14-Nonafluorotetradecylpyridinium
6
F
2
2
ꢁ
CH CH Br), 2.07 (m, 2H, R CH CH –), 3.40 (t, J ¼ 6.8 Hz,
bromide monohydrate (14a). Mp—broad phase transition at
2
2
F
2
2
1
3
2
H, ꢁCH CH Br). C NMR d/ppm 20.3 (t, J ¼ 4.0 Hz), 28.4,
ambient temperature; determination of the onset of this
1
2
2
1
9
2
8.9, 29.3, 29.4, 29.5, 29.5, 31.1 (t, J ¼ 21.9 Hz), 33.1, 33.9.
), ꢁ114.9, ꢁ122.4 (3 ꢂ CF ), ꢁ123.3,
124.1, ꢁ126.7. GC/MS m/z (relative intensity, %) 638 (M ꢁ
F
transition was not possible. H NMR d/ppm 1.2–1.5 (m,
12H, –(CH ) –), 1.60 (m, 2H, R CH CH –), 1.9–2.1 (m, 4H,
NMR d/ppm ꢁ81.4 (CF
3
2
2
6
F
2
2
ꢁ
R CH CH – and ꢁCH CH –py), 3.1 (br s, OH), 5.00 (t, J ¼
F
2
2
2
2
1
, o1), 517 (13), 503 (20), 151 (10), 137 (100), 135 (96).
7.2 Hz, 2H, ꢁCH CH –py), 8.25 (t, J ¼ 6.6 Hz, 2H, H-3,5),
2
2
ꢁ1
IR(KBr) n/cm 2937, 2854, 1246, 1205, 1151.
8.63 (t, J ¼ 7.8 Hz, 1H, H-4), 9.51 (d, J ¼ 6.0 Hz, 2H, H-2,6).
13
C NMR d/ppm 20.00 (t, J ¼ 3.6 Hz), 26.02, 28.97, 28.99,
9.11, 29.18, 29.20, 30.67 (t, J ¼ 22.4 Hz), 31.95, 62.05, 128.58,
2
6
General procedure for pyridinium bromides 13a–c and 14a–c
2
1
9
Bromides (12a–c, 13a–c, 0.1 mol) and anhydrous pyridine (41
ml, 0.5 mol) were refluxed for 10 hours. A white precipitate
was obtained after addition of ether to the cold reaction
mixture. The filtrate was washed thoroughly with ether and
145.07, 145.32. F NMR d/ppm ꢁ81.6 (CF ), ꢁ115.1,
3
ꢁ1
ꢁ125.0, ꢁ126.6. MS m/z (calcd) 438 (438). IR(KBr) n/cm
2930, 2852, 1234, 1214, 1194, 1133. Anal. calcd for
NBr ꢃ H O: C 42.53; H 4.85; N 2.61. Found: C
42.51; H 4.96; N 2.67%.
C
19
H
25
F
9
2
recrystallized twice from acetone. Typical yields were 80–85%
1
(
py ¼ H
5 5
C N ).
1
1,11,12,12,13,13,14,14,15,15,16,16,16-Pentadecafluorohexa-
1
2,12,13,13,14,14,15,15,15-Nonafluoropentadecylpyridinium
decylpyridinium bromide monohydrate (14b). Mp 54.9 1C,
1
bromide monohydrate (13a). Mp 25.5 1C (minor phase transi-
tion), 45.0 1C. H NMR d/ppm 1.2–1.5 (m, 14H, –(CH
73.0 1C. H NMR d/ppm 1.2–1.5 (m, 12H, –(CH
2
)
6
–), 1.60
– and
1
2
)
7
–),
(m, 2H, R CH CH –), 1.9–2.1 (m, 4H, R CH CH
2
F
2
2
F
2
1
.57 (m, 2H, R CH CH –), 1.9–2.1 (m, 4H, R CH CH – and
2
F
2
2
F
2
ꢁCH
2
2
CH
2
–py), 2.5 (br s, OH), 4.99 (t, J ¼ 7.6 Hz, 2H,
–py), 8.20 (t, J ¼ 6.6 Hz, 2H, H-3,5), 8.57 (t, J ¼
ꢁ
ꢁ
CH
2
CH
2
–py), 3.2 (br s, OH), 4.98 (t, J ¼ 7.5 Hz, 2H,
–py), 8.24 (t, J ¼ 6.3 Hz, 2H, H-3,5), 8.63 (t, J ¼
ꢁCH
CH
2
1
3
CH
2
CH
2
7.8 Hz, 1H, H-4), 9.52 (d, J ¼ 5.6 Hz, 2H, H-2,6). C NMR
d/ppm 19.91 (t, J ¼ 3.6 Hz), 25.89, 28.87 (2 ꢂ C), 29.00, 29.06,
29.08, 30.67 (t, J ¼ 22.2 Hz), 31.85, 61.93, 128.41, 145.00,
1
3
7
.8 Hz, 1H, H-4), 9.50 (d, J ¼ 5.7 Hz, 2H, H-2,6). C NMR
d/ppm 19.91 (t, J ¼ 3.7 Hz), 25.93, 28.90, 28.93, 29.02, 29.04,
1
9
2
9.17, 29.24, 30.67 (t, J ¼ 21.8 Hz), 31.82, 61.93, 128.41,
145.11. F NMR d/ppm ꢁ81.5 (CF ), ꢁ115.0, ꢁ122.6,
3
1
9
1
45.00, 145.11. F NMR d/ppm ꢁ81.6 (CF ), ꢁ115.1,
ꢁ123.6, ꢁ124.2, ꢁ126.8. MS m/z (calcd) 538 (538). IR(KBr)
3
This journal is ꢀc the Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2006
New J. Chem., 2006, 30, 944–951 | 949

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ꢁ
1
n/cm 2922, 2843, 1253, 1217, 1175, 1146. Anal. calcd for
13NBr ꢃ H O: C 39.62; H 4.25; N 2.20. Found: C
9.95; H 4.18; N 2.24%.
calculated using GraphPad PRISM V.3.00 (GraphPad Soft-
ware Inc., San Diego, CA).
C
21
H
25
F
2
3
The results from the MTT assay were further confirmed by
Hoffman modulation contrast and fluorescent microscopy
1
1,11,12,12,13,13,14,14,15,15,16,16,17,17,18,18,18-Hepta-
(
epifluorescent inverted microscope T200, Nikon, Japan) ex-
decafluorooctadecylpyridinium bromide monohydrate (14c). Mp
1
8.7 1C. H NMR d/ppm 1.2–1.6 (m, 12H, –(CH
posing morphological changes of the cells treated with various
pyridinium salts. Propidium iodide and YO-PRO-1 (Molecu-
lar Probes, Oregon, USA), were used to distinguish dead or
6
2
)
6
–), 1.58 (m,
CH and
2
H, CH CH –), 1.9–2.1 (m, 4H, R CH
F
R
F
2
2
2
2
–
ꢁ
ꢁ
CH
2
CH
2
–py), 2.7 (br s, OH), 5.01 (t, J ¼ 7.6 Hz, 2H,
–py), 8.21 (t, J ¼ 6.6 Hz, 2H, H-3,5), 8.59 (t, J ¼
39
apoptotic cells from vital living ones (data not shown).
CH
2
CH
2
1
3
7
.8 Hz, 1H, H-4), 9.53 (d, J ¼ 5.6 Hz, 2H, H-2,6). C NMR
Haemolytic activity
d/ppm 20.15 (t, J ¼ 3.6 Hz), 26.15, 29.13 (2 ꢂ C), 29.27, 29.33,
Rabbit red blood cells (2% in PBS) were used to perform
standard haemolytic tests in both PBS and PBS containing
2
9.35, 30.90 (t, J ¼ 21.75 Hz), 32.12, 62.14, 128.68, 145.43,
19
1
45.58. F NMR d/ppm ꢁ81.4 (CF ), ꢁ115.0, ꢁ122.5 (3 ꢂ
3
2
0% fetal bovine serum. Concentration ranges of tested
CF
IR(KBr) n/cm 2921, 2853, 1253, 1209, 1169, 1139. Anal.
calcd for C23 O: C 37.50; H 3.6; N 1.90. Found:
17NBr ꢃ H
C 37.49; H 3.60; N 1.88%.
2
), ꢁ123.1, ꢁ124.1, ꢁ126.8. MS m/z (calcd) 638 (638).
compounds were from 1 mM to 1 mM. The released haemo-
globin was quantified using a well-plate spectrophotometer
reader iEMS MF (Labsystems, Turku, Finland) at 540 nm
after two hours incubation of red cells with a particular
compound at 37 1C. Data were expressed as the lowest
concentration of surfactants causing haemolysis.
ꢁ1
H
25
F
2
Assessment of cytotoxicity
Cancer cell lines
Acknowledgements
The A2780 ovarian carcinoma cell line (ECACC) was selected
from a panel of cancer cell lines used for testing in our
We thank Mr John Snyder (Department of Chemistry, The
University of Iowa) for helpful discussions of the NMR
experiments and Dr Paul Bummer (College of Pharmacy,
University of Kentucky) for access to the DSC instrument.
This material is based upon work supported by the National
Institute of Environmental Health Sciences (ES12475) and the
National Science Foundation (NIRT 0210517). This work was
also supported by grants from the Ministry of Agriculture of
the Czech Republic (grant No. MZE-0002716201) and NAZV
QF 3115. Its contents are solely the responsibility of the
authors and do not necessarily represent the official views of
the funding agencies.
2
7
laboratory. The cell line was grown in RPMI 1640 medium
Sigma, Czech Republic) supplemented with 10% of fetal calf
serum (Gibco, Czech Republic), 40 IU of insulin per 100 ml of
(
ꢁ
1
ꢁ1
medium, 50 mg ml penicillin, 50 mg ml streptomycin, 100
ꢁ
1
ꢁ1
mg ml neomycin, and 300 mg ml L-glutamine as reported
previously. Cultures were maintained in a humidified incu-
2
7
bator at 37 1C and 5% CO
2
.
MTT-based cytotoxicity test
3
7,38
The MTT assay
was used to assess the cytotoxicity of the
pyridinium salts in cells in the exponential growth phase. In
short, cells were seeded on 96-well flat-bottom microplates at
4
the density 2.5–3.0 ꢂ 10 per ml, 100 ml per well, and allowed
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