8222
K. Musilek et al. / Bioorg. Med. Chem. 16 (2008) 8218–8223
2H, J = 6.0 Hz, H-3,5), 7.61–7.51 (m, 2H, Ph), 7.50–7.38 (m, 3H, Ph),
5.88 (s, 2H, –CH2–). 13C NMR (75 MHz, DMSO d6): d 148.69, 145.01,
144.94, 134.22, 129.25, 129.13, 128.74, 124.32, 62.58. EA: calcu-
lated 53.26% C, 4.47% H, 9.56% N; found 52.70% C, 4.86% H, 9.21%
N. ESI–MS: m/z 213.1 [M]+ (calculated for [C13H13N2O]+ 213.10).
5.2.18. 1-(3-Phenylpropyl)-4-hydroxyiminomethylpyridinium-
bromide (23)
TLC Rf 0.73. M.p. 161–163 °C. 1H NMR (300 MHz, DMSO d6): d
9.10 (d, 2H, J = 6.0 Hz, H-2,6), 8.45 (s, 1H, ACH@NOH), 8.22 (d,
1H, J = 6.0 Hz, H-3,5), 7.32–7.12 (m, 5H, Ph), 4.65 (t, 2H,
J = 7.3 Hz, N–CH2–), 2.66 (t, 2H, J = 7.3 Hz, N–CH2–CH2–), 2.33-
2.17 (m, 2H, Ph–CH2–). 13C NMR (75 MHz, DMSO d6): d 148.22,
145.00, 144.95, 140.22, 128.32, 128.14, 126.01, 123.90, 60.02,
31.88, 31.47. EA: calculated 56.09% C, 5.33% H, 8.72% N; found
56.66% C, 5.94% H, 9.04% N. ESI–MS: m/z 241.1 [M]+ (calculated
for [C15H17N2O]+ 241.13).
5.2.13. 1-(2-Phenylethyl)-2-hydroxyiminomethylpyridinium-
bromide (18)
TLC Rf 0.66. M.p. 164–166 °C. 1H NMR (300 MHz, DMSO d6): d
8.90 (d, 1H, J = 6.0 Hz, H-6), 8.73 (s, 1H, ACH@NOH), 8.59–8.48
(m, 1H, H-5), 8.36 (d, 1H, J = 8.1 Hz, H-3), 8.10–7.96 (m, 1H, H-4),
7.39–7.10 (m, 5H, Ph), 5.04 (t, 2H, J = 7.3 Hz, N–CH2–), 3.17 (t, 2H,
J = 7.3 Hz, Ph–CH2–). 13C NMR (75 MHz, DMSO d6): d 146.82,
145.95, 145.28, 141.37, 135.69, 128.91, 128.52, 127.12, 125.82,
58.46. EA: calculated 54.74% C, 4.92% H, 9.12% N; found 54.14% C,
4.98% H, 9.21% N. ESI–MS: m/z 227.1 [M]+ (calculated for
[C14H15N2O]+ 227.12).
5.3. In vitro assay
In vitro testing of reactivators has been described in detail.28
Briefly, the 10% rat brain homogenate in distilled water was used
as a source of AChE. The brain homogenate (0.5 mL) was mixed
with 20 lL of isopropanol solution of tabun (tabun, O-ethyl-N,N-
5.2.14. 1-(2-Phenylethyl)-3-hydroxyiminomethylpyridinium-
bromide (19)
dimethylphosphoramidocyanidate; obtained from the Military
facility Brno, 95% purity) or paraoxon (O,O-diethyl-O-(4-nitro-
phenyl)phosphate, analytical standard 99.2% from Sigma–Al-
drich) and distilled water (0.5 mL). The mixture was incubated
at 25 °C for 30 min to achieve 95% inhibition of AChE. The mix-
ture was filled in assay vessel to the volume 23 mL with distilled
water and sodium chloride (3 M; 2.5 mL) was added. Finally,
2 mL of acetylcholine iodide (0.02 M; substrate for enzymatic
reaction) was added. The enzyme activity (analyzed by potentio-
metric titration of decomposed acetylcholine iodide) was mea-
sured at pH 7.6 and 25 °C using an autotitrator Titrando 842
(Metrohm, Switzerland).
TLC Rf 0.63. M.p. 215–216 °C. 1H NMR (300 MHz, DMSO d6): d
9.30 (s, 1H, H-2), 9.04 (d, 1H, J = 6.2 Hz, H-6), 8.71 (d, 1H,
J = 8.2 Hz, H-4), 8.32 (s, 1H, ACH@NOH), 8.17–8.09 (m, 1H, H-5),
7.35–7.20 (m, 5H, Ph), 4.91 (t, 2H, J = 7.3 Hz, N–CH2–), 3.30 (t, 2H,
J = 7.3 Hz, Ph–CH2–). 13C NMR (75 MHz, DMSO d6): d 144.28,
143.11, 142.39, 141.55, 136.00, 133.16, 128.84, 128.53, 127.83,
127.00, 61.62, 36.26. EA: calculated 54.74% C, 4.92% H, 9.12% N;
found 55.30% C, 5.19% H, 9.44% N. ESI–MS: m/z 227.1 [M]+ (calcu-
lated for [C14H15N2O]+ 227.12).
5.2.15. 1-(2-Phenylethyl)-4-hydroxyiminomethylpyridinium-
bromide (20)
The same procedure was repeated with rat brain homogenate,
which was 30 min incubated with tabun or paraoxon and further
treated for 10 min with an aqueous solution of reactivator
(0.2 mL – replacing same amount of distilled water). Activities of
intact AChE (a0), inhibited AChE (ai) and reactivated AChE (ar) were
deduced from the amount of NaOH solution (0.01 M) versus time;
NaOH reacted with acetic acid released from decomposed acetyl-
choline iodide. The percentage of reactivation (%) was calculated
from the measured data according to the formula
TLC Rf 0.67. M.p. 220–222 °C. 1H NMR (300 MHz, DMSO d6): d
9.03 (d, 2H, J = 6.2 Hz, H-2,6), 8.42 (s, 1H, ACH@NOH), 8.20 (d,
2H, J = 6.2 Hz, H-3,5), 7.34–7.20 (m, 5H, Ph), 4.86 (t, 2H,
J = 7.3 Hz, N–CH2–), 3.27 (t, 2H, J = 7.3 Hz, Ph–CH2–). 13C NMR
(75 MHz, DMSO d6): d 148.33, 144.98, 144.90, 136.05, 128.82,
128.53, 126.98, 123.74, 60.88, 36.28. EA: calculated 54.74% C,
4.92% H, 9.12% N; found 54.71% C, 5.19% H, 9.33% N. ESI–MS: m/z
227.1 [M]+ (calculated for [C14H15N2O]+ 227.12).
ꢀ
ꢁ
a0 ꢀ ar
a0 ꢀ ai
x ¼ 1 ꢀ
ꢁ 100 ð%Þ:
5.2.16. 1-(3-Phenylpropyl)-2-hydroxyiminomethylpyridinium-
bromide (21)
TLC Rf 0.73. M.p. 129–131 °C. 1H NMR (300 MHz, DMSO d6): d
9.10 (d, 1H, J = 6.0 Hz, H-6), 8.76 (s, 1H, ACH@NOH), 8.59–8.50
(m, 1H, H-5), 8.38 (d, 1H, J = 8.1 Hz, H-3), 8.13–8.04 (m, 1H, H-4),
7.33–7.15 (m, 5H, Ph), 4.78 (t, 2H, J = 7.3 Hz, N–CH2–), 2.71 (t,
2H, J = 7.3 Hz, N–CH2–CH2–), 2.20–2.04 (m, 2H, Ph–CH2–). 13C
NMR (75 MHz, DMSO d6): d 146.76, 145.92, 145.08, 141.39,
140.27, 128.35, 128.11, 127.37, 126.07, 125.73, 57.59, 31.77,
31.35. EA: calculated 56.09% C, 5.33% H, 8.72% N; found 55.84% C,
5.43% H, 8.83% N. ESI–MS: m/z 241.1 [M]+ (calculated for
[C15H17N2O]+ 241.13).
Acknowledgements
The authors express their appreciation to Mrs. M. Hrabinova
and Ms. Petra Hanusova for their technical assistance. The work
was supported by Grant Agency of Ministry of Education, Youth
and Sports (Czech Republic) – Grant No. ME865.
References and notes
1. Giacobini, E. In Cholinesterases and Cholinesterase Inhibitors; Giacobini, E., Ed.;
Martin Dunitz: London, 2000; pp 1–226.
2. Giacobini, E. Pharmacol. Res. 2004, 50, 433.
3. Marrs, T. C. Pharmacol. Therap. 1993, 58, 51.
4. Bajgar, J. Adv. Clin. Chem. 2004, 38, 151.
5. Saxena, A.; Sun, W.; Luo, C.; Myers, T. M.; Koplovitz, I.; Lenz, D. E.; Doctor, B. P. J.
Mol. Neurosci. 2006, 30, 145.
6. Hagedorn, I.; Gündel, W. H.; Schoene, K. Arzneimittelforschung 1969, 19, 603.
7. Lüttringhaus, A.; Hagedorn, I. Arzneimittelforschung 1964, 14, 1.
8. Poziomek, E. J.; Hackley, B. E.; Steinberg, G. M. J. Org. Chem. 1958, 23, 714.
9. Ekstrom, F.; Akfur, C.; Tunemalm, A. K.; Lundberg, S. Biochemistry 2006, 45, 74.
10. Ekstrom, F.; Pang, Y. P.; Boman, M.; Artursson, E.; Akfur, C.; Borjegren, S.
Biochem. Pharmacol. 2006, 72, 597.
11. de Jong, L. P. A.; Benschop, H. P.; van den Berg, G. R.; Wolring, G. Z.; de Korte, D.
C. Eur. J. Med. Chem. 1981, 16, 257.
12. Odzak, R.; Calic, M.; Hrenar, T.; Primozic, I.; Kovarik, Z. Toxicology 2007, 233, 85.
13. Okuno, S.; Sakurada, K.; Ohta, H.; Ikegaya, H.; Kazui, Y.; Akutsu, T.; Takatori, T.;
Iwadate, K. Toxicol. Appl. Pharmacol. 2008, 227, 8.
5.2.17. 1-(3-Phenylpropyl)-3-hydroxyiminomethylpyridinium-
bromide (22)
TLC Rf 0.71. M.p. 143–145 °C. 1H NMR (300 MHz, DMSO d6): d
9.33 (s, 1H, H-2), 9.13 (d, 1H, J = 6.0 Hz, H-6), 8.72 (d, 1H,
J = 8.1 Hz, H-4), 8.35 (s, 1H, ACH@NOH), 8.19–8.11 (m, 1H, H-5),
7.32–7.13 (m, 5H, Ph), 4.71 (t, 2H, J = 7.3 Hz, N–CH2–), 2.67 (t,
2H, J = 7.3 Hz, N–CH2–CH2–), 2.37–2.21 (m, 2H, Ph–CH2–). 13C
NMR (75 MHz, DMSO d6): d 144.37, 143.18, 142.60, 141.18,
140.21, 133.28, 128.30, 128.13, 127.96, 126.02, 60.79, 31.82,
31.49. EA: calculated 56.09% C, 5.33% H, 8.72% N; found 54.52% C,
4.98% H, 8.52% N. ESI–MS: m/z 241.1 [M]+ (calculated for
[C15H17N2O]+ 241.13).