N-p-Methoxybenzyl tetrahydropyranyl dimeric pyridinium iod-
ide 8. To a solution of 5 (0.35 g, 1.20 mmol) and 7 (0.57 g, 1.10
mmol) in CH3CN (10 cm3), 0.18 g of potassium iodide were
added. The mixture was heated at reflux for 20 hours, cooled to
room temperature and evaporated. The residue was washed
with hexane/EtOAc 9 : 1 to remove the excess monomer 5 and
then submitted to FC (Al2O3, hexane/EtOAc 7 : 3, CHCl3/
CH3OH 94 : 6, CH3OH) to yield the dimer 8 (0.75 g, 80%);
δH(300 MHz, CDCl3 ϩ CD3OD): 8.90 (4H, m, 2Ј-H, 6Ј-H, 2Љ-
H, 6Љ-H), 8.40 (2H, m, 4Ј-H, 4Љ-H), 8.05–7.95 (2H, m, 5Ј-H, 5Љ-
H), 7.52 (2H, d, J 8.7, Ar), 6.91 (2H, d, J 8.7, Ar), 5.97 (2H, s,
ArCH2), 4.73 (3H, m, 2ٞ-H and 7Ј-H2), 3.70 (3H, s, OMe),
3.80–3.20 (4H, series of m, 1-H2 and 6ٞ-H2), 2.80 (4H, m, 8-H2,
14Ј-H2), 1.90–1.30 (series of m, 2-H2–7-H2, 8Ј-H2, 13Ј-H2, and
3ٞ-H–5ٞ-H); δC (75 MHz, CDCl3 ϩ CD3OD): 160.64 (s, Ar),
145.32, 144.91, 144.79, 144.29, 144.28, 144.24, 144.07, 143.70
(pyridinium), 131.12 (2C, d, Ar), 127.91 and 127.77 (all d,
pyridinium), 124.46 (s, Ar), 114.79 (2 C, d, Ar), 98.83 (d, C-2ٞ)
63.80, 62.96, 62.36, 62.09 (all t, C-1, C-7Ј, C-6ٞ, ArCH2), 55.28
(q, OMe), 32.80–25.35 (all t, C-2–C-8, C-8Ј–C-14Ј, C-3Љ–C-5Љ);
ESI-MS : m/z 729 ([M Ϫ I]ϩ).
0.29 mmol) in CH3CN (20 cm3) was refluxed with stirring for 20
hours, and then cooled to room temperature. Evaporation of
the solvent gave a crude mixture from which a slight excess of
dimer 10 was taken away by washing with CHCl3/MeOH 95 : 5.
The remaining residue was a quite pure tetramer 11 (0.29 g,
75%). A small amount of 11 has been purified by FC (-NH2
stationary phase, H2O/CH3CN gradient elution) for biological
testing. δH (300 MHz, CD3OD): 9.03 (4H, s), 8.87 (4H, m), 8.46
(4H, m), 8.00 (4H, m), 7.53 (2H, d, J 8.7, Ar), 6.99 (2H, d, J 8.7,
Ar), 5.78 (2H, s, ArCH2), 4.65 (7H, m, 3 × NCH2, OCHO), 3.80
(3H, s, OMe), 3.80–3.30 (4H, series of m) 2.89 (8H, m, 4 ×
pyridinium-CH2), 2.10–1.30 (series of m); ESI-MS: m/z 1513
([M Ϫ I ϩ NaI]ϩ), 1363 ([M Ϫ I]ϩ).
Hydroxyl dimeric pyridinium iodide 12. To a solution of 10
(0.018 g, 0.03 mmol) in MeOH (1 cm3), p-toluenesulfonic acid
monohydrate (0.008 g) was added. The resulting mixture was
stirred at room temperature for 2 h and then evaporated. Potas-
sium iodide in excess was added to the residue in acetonitrile
(3 ml) and the mixture was stirred for 1 h. After that it was
filtered and the filtrate evaporated, to leave a residue that was
subjected to FC (-NH2 stationary phase, H2O/CH3CN) to give
12 (0.013 g, 86%). δH (300 MHz, CDCl3): 8.90 (2H, m), 8.42
(3H, m), 8.00 (1H, m), 7.48 (1H, m), 7.20 (1H, m), 4.69 (2H, t,
J 7.6, CH2N), 3.49 (2H, t, J 6.7, CH2OH), 2.78 (2H, t, J 7.6,
pyridinium-CH2), 2.46 (2H, t, J 7.6, pyridine-CH2), 1.90–1.30
(24H, series of m); ESI-MS: m/z 397 ([M Ϫ I]ϩ). MS/MS (397):
208, 190.
N-p-Methoxybenzyl dimeric pyridinium iodide 9. Phosphorus
tribromide (0.15 cm3, 1.65 mmol) was added dropwise to a solu-
tion of dimer 8 (0.35 g, 0.41 mmol) in CHCl3/CH3CN 1 : 1 (10
cm3) at 0 ЊC under nitrogen. The solution was stirred for 15
minutes at 0 ЊC, heated under reflux for 1 hour, cooled to room
temperature, filtered and quenched with sat. aq. NaHCO3. The
residue from evaporation was subjected to FC (silica gel,
EtOAc/hexane 2 : 1, then CH3OH), to give the corresponding
pure bromide (0.28 g); δH(300 MHz, CDCl3 ϩ CD3OD): 8.90
(4H, m, 2Ј-H, 6Ј-H, 2Љ-H, 6Љ-H), 8.40 (2H, m, 4Ј-H, 4Љ-H),
7.98 (2H, m, 5Ј-H, 5Љ-H), 7.58 (2H, d, J 8.7, Ar), 6.95 (2H, d,
J 8.7, Ar), 5.94 (2H, s, ArCH2), 4.75 (3H, m, 2ٞ-H and 7Ј-H2),
3.81 (3H, s, OMe), 3.38 (2H, t, J 6.8, 1-H2), 2.84 (4H, m, 8-H2,
14Ј-H2), 1.90–1.24 (series of m, 2-H2–7-H2, 8Ј-H2–13Ј-H2);
ESI-MS : m/z 707/709 ([M Ϫ I]ϩ); MS/MS (707/709): 459/461.
Potassium iodide (0.06 g, 0.38 mmol) was added to a solution
of bromide (0.27 g, 0.32 mmol) in CH3CN (5 cm3). The mixture
was heated under reflux for 20 hours under stirring, and then
cooled to room temperature. The residue from evaporation of
the solvent was subjected to FC (CN stationary phase, from
H2O/CH3CN 97 : 3, to CH3CN) to give iodide 9 (0.25 g, 74% of
overall yield from 8); δH(300 MHz, CDCl3 ϩ CD3OD): 8.90
(4H, m, 2Ј-H, 6Ј-H, 2ٞ-H, 6ٞ-H), 8.42 (2H, m, 4Ј-H, 4Љ-H),
7.96 (2H, m, 5Ј-H, 5Љ-H), 7.60 (2H, d, J 8.7, Ar), 6.95 (2H, d,
J 8.7, Ar), 5.92 (2H, s, ArCH2), 4.75 (3H, m, 2ٞ-H and 7Ј-H2),
3.82 (3H, s, OMe), 3.17 (2H, t, J 6.8, 1-H2), 2.85 (4H, m,
8-H2, 14Ј-H2), 1.95–1.24 (series of m, 2-H2–7-H2, 8Ј-H2–13Ј-
H2); ESI-MS : m/z 755 ([M Ϫ I]ϩ).
Tetrameric pyridinium iodide 13. During the conversion of
the tetramer 11 to the corresponding iodide by the same treat-
ment used to obtain dimer 9 from 8, accidental evaporation of
the reaction mixture to dryness caused a partial N-PMB depro-
tection, giving the minor compound 13 together with the
expected iodide.
FC (-NH2 stationary phase, H2O/CH3CN gradient elution)
then furnished the major product in good yield and a small
amount of the less polar tetramer 13 (10.8 mg), but sufficient to
be tested for biological activities. δH (300 MHz, CD3OD ϩ
CDCl3): 9.0–8.05 (series of m, pyridinium and pyridine), 7.46
(1H, br d, J 7.6, pyridine), 7.18 (1H, m, pyridine), 4.78 (6H, m,
N CH2), 2.80 (6H, m, pyridinium-CH2), 3.15 (2H, t, J 6.9,
CH2I), 2.56 (2H, m, pyridine-CH2), 2.00–1.30 (series of m);
ESI-MS: m/z 1141 ([M Ϫ I]ϩ).
Bioassays
Synthetic and natural alkylpyridinium compounds. For bio-
logical tests, the stock solutions (10 mg mlϪ1) in deionized water
(compounds 4ؒHCl and 14ؒHCl), methanol (compounds 4–7,
11, 12 and 14), or ethanol (compound 13) were prepared and
kept at 4 ЊC prior to use. Poly-APS compounds (1) were isolated
from the marine sponge Reniera sarai by a standard procedure.1
For biological tests, a stock solution (10 mg mlϪ1) was prepared
in deionized water and kept at 4 ЊC until use. Since poly-APS
compounds are a mixture of two polymers, the molar concen-
tration calculations were estimated assuming a 1 : 1 ratio
between them, giving a molecular weight of 12200 Da.
Tetrahydropyranyl dimeric pyridinium iodide 10. A solution of
dimer 8 (0.38 g, 0.44 mmol) and 3-methylpyridine (4, 1.0 cm3,
10.6 mmol) was heated under reflux for 16 hours with stirring,
then cooled to room temperature. The residue from evaporation
was subjected to FC (-NH2 stationary phase, from H2O/
CH3CN 97 : 3 to CH3CN), to give 10 (0.20 g, 75%); δH(300
MHz, CDCl3): 8.90 (2H, m, 2Ј-H, 6Ј-H), 8.42 (3H, m), 8.00
(1H, m), 7.46 (1H, m), 7.19 (1H, m), 4.73 (3H, m, 2ٞ-H and 7Ј-
H2), 4.56 (1H, m, 2ٞ-H), 3.80–3.20 (4H, series of m, 1-H2 and
6ٞ-H2), 2.86 (2H, m, 8-H2), 2.56 (2H, m, 14Ј-H2), 1.90–1.30
(series of m, 2-H2–7-H2, 8Ј-H2–13Ј-H2 and 3ٞ-H2–5ٞ-H2); δC (75
MHz, CDCl3 ϩ CD3OD): 146.96, 146.70,146.12, 145.80,
144.96, 144.79, 137.92, 137.50, 128.35, 126.61 (C-2Ј–C-6Ј and
C-2Љ–C-6Љ); 98.85 (d, C-2ٞ), 63.26, 62.36, 61.75 (all t, C-1, C-7Ј,
C-6ٞ), 32.35–25.30 (all t, C-2–C-8, C-8Ј–C-14Ј, C-3Љ–C-5Љ);
ESI-MS: m/z 481 ([M Ϫ I]ϩ).
Estimation of biological activities. Antimicrobial activity. The
following bacterial strains, obtained from the local collection at
the Department of Biology, University of Ljubljana, were used:
Staphylococcus aureus, Streptococcus foecalis, Micrococcus
luteus, Bacillus subtilis, Escherichia coli, Proteus vulgaris, Kleb-
siella pneumoniae, Pseudomonas aeruginosa, and Salmonella
typhimurium. Activities were evaluated using the standard agar
diffusion test. Bacteria were allowed to grow overnight and
their concentration then determined. Bacterial culture was
incorporated in Lauria Broth nutrient agar previously cooled to
42 ЊC. The final concentration of bacteria was approximately
5 × 105 CFU mlϪ1 (CFU: colony forming unit). Twenty milli-
N-p-Methoxybenzyl tetrahydropyranyl tetrameric pyridinium
iodide 11. A solution of 9 (0.23 g, 0.26 mmol) and 10 (0.18 g,
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 3 6 8 – 1 3 7 5
1373