Preparation and in vitro evaluation of monoquaternary inhibitors of brain cholinesterases

Article abstract of DOI:10.2174/157017809789124876

Acetylcholinesterase inhibitors are currently of high interest due to the many reasons. Among them, Alzheimer's disease drugs are of great interest. In this study, eleven monoquaternary pyridinium salts substituted by different groups (electron donors and

Full text of DOI:10.2174/157017809789124876

5
00
Letters in Organic Chemistry, 2009, 6, 500-503
Preparation and In Vitro Evaluation of Monoquaternary Inhibitors of
Brain Cholinesterases
1
,2
3
3
3
1,4
1
Daniel Jun , Martin Paar , Jiri Binder , Jan Marek , Miroslav Pohanka , Petr Stodulka and Kamil
Kuca*
,1,2,5
1
Center of Advanced Studies, Faculty of Military Health Sciences, University of Defence, Czech Republic
Department of Toxicology, Faculty of Military Health Sciences, University of Defence, Czech Republic
Department of Pharmaceutical Chemistry and Drug Control, Faculty of Pharmacy in Hradec Kralove, Charles
2
3
University in Prague, Hradec Kralove, Czech Republic
4
Centre of Biological Defense, Techonin, Central Military Institute of Health, Czech Republic
5
Department of Chemistry, Faculty of Sciences, J.E. Purkinje University, Horeni 13, 400 96, Usti nad Labem, Czech
Republic
Received March 04, 2008: Revised June 08, 2009: Accepted June 17, 2009
Abstract: Acetylcholinesterase inhibitors are currently of high interest due to the many reasons. Among them,
Alzheimer´s disease drugs are of great interest. In this study, eleven monoquaternary pyridinium salts substituted by
different groups (electron donors and electron acceptors) in position four were prepared. Then, their anticholinesterase
activity was tested. As observed, their IC50s ranged from 33 μM to 3981 μM. Moreover, probability of blood-brain
barrier penetration was predicted.
Keywords: Quaternary ammonium salts, pyridinium salts, inhibitor, cholinesterase, Alzheimer´s disease, prophylaxis.
INTRODUCTION
are of very high importance. Thanks to it, quaternary
ammonium salts could be considered as a potential treatment
of Alzheimer´s disease or at least as its supplement.
Monoquaternary and bisquaternary pyridinium salts are
known as inhibitors of the enzyme acetylcholinesterase
(
[
AChE; EC 3.1.1.7) - enzyme involved in neurotransmission
1]. For example, some of them were investigated in the past
Due to the above-mentioned findings, in this study, we
have focused our attention to the preparation of
monoquaternary compounds. Choice of monoquaternary
compounds is based on the higher percent ability of these
compounds to penetrate the BBB, if compared with
bisquaternary substances. We have prepared eleven
quaternary pyridinium derivatives differing only in the
substituent presented in the position four at the pyridinium
ring. Then, we have tested their ability to inhibit brain
cholinesterases.
as prophylactic agent against nerve agents [2,3].
Bisquaternary compound SAD-128 is probably the most
known member of this family. Promising prophylactic effect
in case of sarin, soman and tabun intoxications was obtained
[
4-6]. Its effect is caused by the competitive inhibition of
AChE. Due to enzyme inhibition, the second inhibitor (e.g.
nerve agent) could not enter the cavity of the enzyme and
further inhibition of AChE by irreversible inhibitor is
impossible [1,4-8].
Other such compounds are at present considered as
potential treatment of Alzheimer´s disease [7,9-11]. In this
sense, permeability of the blood-brain barrier (BBB) by
quaternary compounds is currently well discussed [12]. In
MATERIAL AND METHODS
Chemicals
All chemicals used in this study were purchased from
Sigma-Aldrich (Czech Republic). Synthesis of all
cholinesterases inhibitors was done according to the Scheme
2
003, Sakurada et al. found that monoquaternary AChE
reactivator - pralidoxime is able to penetrate BBB in 10 %
13]. More recently, Lorke et al. found that also
bisquaternary compounds are able to penetrate BBB in only
.5 % [14]. There were published also several papers focused
[
1
. In all cases, appropriate substituted pyridine derivative
was dissolved in acetone and treated with the excess of
methyliodide (5x) to produce crystalline monopyridinium
salts which were filtered and subsequently recrystalized from
n-butanol. Melting points (Boetius, Germany), NMR spectra
1
on the prediction of lipophilicity (or hydrophilicity) of ionic
compounds and prediction of their BBB penetration [15-17].
These findings (BBB penetration of quaternary compounds)
(
Varian Gemini 300, Palo Alto CA, USA) of all prepared
compounds are listed below:
*
Address correspondence to this author at the Center of Advanced Studies,
Faculty of Military Health Sciences, University of Defence, Trebesska
575, Hradec Kralove, Czech Republic; Tel: +420973251523; Fax:
420495518094; E-mail: kucakam@pmfhk.cz; kucakam@gmail.com
4
-[(hydroximino)methyl]-1-methylpyridinium iodide (1)
1
yield 45%; mp 179-180 ˚C; H NMR (300 MHz, DMSO) d
ppm 9.07 (d, J = 6.00 Hz, 2H), 8.43 (s, 1H), 8.23 (d, J = 6.00
1
+
1
570-1786/09 $55.00+.00
© 2009 Bentham Science Publishers Ltd.

Preparation and In Vitro Evaluation of Monoquaternary Inhibitors
Letters in Organic Chemistry, 2009, Vol. 6, No. 6
501
1
3
Hz, 2H); 4.60 (s, 3H). C NMR (75 MHz, DMSO d6): ꢀ
(
4-hydroxymethyl-1-methylpyridinium iodide (11) yield
82%; mp 130 ˚C; H NMR (300 MHz, DMSO) d ppm 9.01
1
ppm) 163.32, 147.73, 146.42, 125.31, 48.00.
(
4
1
d, J = 6.61 Hz, 2H), 8.42 (d, J = 6.71 Hz, 2H), 4.81 (s, 2H)
.48 (s, 3H). C NMR (75 MHz, DMSO d6): ꢀ (ppm)
62.01, 144.76, 123.85, 61.06, 47.24.
R
R
13
+
CH3 I
I-
N+
N
In Vitro Experiments
CH3
Rat brain cholinesterases were used as the source of the
enzyme. As it is generally known, percentage ratio of
Scheme 1.
cholinesterase
butyrylcholinesterase (BuChE; 3.1.1.8) in rat brain is 80% to
0% [18]. Preparation of rat brain homogenates was as
follows: Rats were obtained from BioTest Konarovice
Czech Republic). They were decapitated, brains were
withdrawn and washed with a saline. Then, brains were
homogenized in distilled water (10% w/v) by the use of an
Ultra-Turrax instrument (Janke–Kunkel, Germany) at 20.000
r.p.m. for 1 min. Aliquots (2 ml) of the homogenate were
stored at -35 °C in a freezer. They were thawed immediately
prior the use.
activities
between
AChE
and
4
-carbamoyl-1-methylpyridinium iodide (2) yield 68%;
1
mp 269-270 ˚C; H NMR (300 MHz, DMSO) d ppm 9.15 (d,
H, J = 6.0), 8.65 (s, 1H), 8.39 (d, 2H, J = 6.0), 8.26 (s, 1H),
.39 (s, 3H). C NMR (75 MHz, DMSO d6): ꢀ (ppm)
63.32, 147.71, 146.42, 125.31, 48.03.
2
2
4
1
13
(
1
-methylpyridinium iodide (3) yield 71%; mp 117-118
1
˚
2
(
1
C; H NMR (300 MHz, DMSO) d ppm 8.98 (d, J = 5.57 Hz,
H), 8.57 (t, J = 7.82 Hz, 1H), 8.12 (t, J = 7.09 Hz, 2H), 4.34
1
3
s, 3H). C NMR (75 MHz, DMSO d6): ꢀ (ppm) 146.25,
45.75, 128.37, 48.67.
4
-cyano-1-methylpyridinium iodide (4) yield 48%; mp
In vitro inhibition test was conducted as described earlier
[7]. Solution of the prepared monoquaternary pyridinium
salts with appropriate concentrations (concentration range
from 10 to 10 M; 500 μl) was added to the rat brain
homogenate (500 μl; 10 w/v), solution of sodium chloride (3
M; 2.5 ml) and water (20 ml). Then a solution of
acetylcholine iodide (0.02 M; 2.0 ml) was added (= starting
1
1
98 ˚C; H NMR (300 MHz, DMSO) d ppm 9.26 (d, J = 6.62
1
3
Hz, 2H), 8.67 (d, J = 6.33 Hz, 2H), 4.40 (s, 3H). C NMR
75 MHz, DMSO d6): ꢀ (ppm) 146.88, 130.38, 114.73,
8.91.
-
7
-1
(
4
4
-(amidoxime)-1-methylpyridinium iodide (5) yield 67%;
1
mp 197 ˚C; H NMR (300 MHz, DMSO) d ppm 10.90 (s,
H), 8.93 (d, J = 6.84 Hz, 2H), 8.26 (d, J = 6.93 Hz, 2H),
.41 (s, 2H), 4.30 (s, 3H). C NMR (75 MHz, DMSO d6): ꢀ
ppm) 147.36, 146.94, 145.26, 122.33, 47.34.
of the enzyme reaction). The enzyme activity (a
immediately determined using automatic titrator RTS 822
Radiometer, Denmark). Values of IC50 for tested inhibitors
I
) was
1
6
(
13
(
were calculated from the enzyme activities at appropriate
inhibitors concentrations using Graph Pad Prism v. 4 for
Windows (GraphPad Software, San Diego, California,
USA).
4
-(dimethylamino)-1-methylpyridinium iodide (6) yield
1
9
8
8%; mp 248 ˚C; H NMR (300 MHz, DMSO) d ppm 8.22-
.18 (m, 2H), 7.03-6.99 (m, 2H), 3.89 (s, 3H), 3.16 (s, 6H).
C NMR (75 MHz, DMSO d6): ꢀ (ppm) 146.28, 142.76,
07.39, 43.92.
1
3
1
Physico-Chemical Properties Calculations
4
-benzyl-1-methylpyridinium iodide (7) yield 56%; mp
1
The partition coefficients (MiLogP), molecular polar
surface area (TPSA) and molecular volume (Vol) of
synthesized substances were calculated online using the
Molinspiration site (Molinspiration, Bratislava, Slovak
Republic) [19].
1
81 ˚C; H NMR (300 MHz, DMSO) d ppm 9.19 (d, J = 6.57
Hz, 2H), 8.34 (d, J = 6.45 Hz, 2H), 7.63 (t, J = 7.82 Hz, 2H),
.85-7.81 (m, 2H), 7.79 (m, 1H), 4.44 (s, 3H), 3.33 (s, 2H).
C NMR (75 MHz, DMSO d
7
1
3
6
): ꢀ (ppm) 151.19, 146.40,
1
34.75, 134.11, 130.16, 129.03, 127.23, 126.39, 48.11.
4
-phenyl-1-methylpyridinium iodide (8) yield 81%; mp
1
1
69 ˚C; H NMR (300 MHz, DMSO) d ppm 9.00 (d, J = 7.06
RESULTS
Hz, 2H), 8.50 (d, J = 7.06 Hz, 2H), 8.09-8.04 (m, 2H), 7.66
All newly prepared compounds were synthesized in
satisfactory yields (range from 45 % to 98 %). The highest
yield was reached for compound 6. All in vitro results are
summarized in Table 1. The lowest values of IC50
characterizing the highest inhibition potency was achieved
for compound 6 having dimethylamino group at the position
four at the pyridinium ring. On the contrary, no inhibition
(
t, J = 2.09 Hz, 2H),7.64 (d, J = 1.80 Hz, 1H), 4.32 (s, 3H).
1
3
C NMR (75 MHz, DMSO d
6
): ꢀ (ppm) 154.22, 145.55,
1
33.50, 132.02, 129.65, 128.00, 124.05, 47.03.
4
-acetyl-1-methylpyridinium iodide (9) yield 66%; mp
1
1
79 ˚C; H NMR (300 MHz, DMSO) d ppm 9.17 (d, J = 6.61
Hz, 2H), 8.46 (d, J = 6.71 Hz, 2H), 4.40 (s, 3H), 2.73 (s, 3H).
1
3
C NMR (75 MHz, DMSO d6): ꢀ (ppm) 195.63, 147.95,
2
was achieved for compound 11 with CH OH group in the
1
46.96, 125.42, 48.15, 27.41.
position four at the pyridinium ring. All other prepared
compounds are considered as medium to weak inhibitors of
brain cholinesterases.
4
-tert-butyl-1-methylpyridinium iodide (10) yield 80%,
1
mp 123-124 ˚C; H NMR (300 MHz, DMSO) d ppm 8.87 (d,
J = 7.08 Hz, 2H), 8.14 (d, J = 7.01 Hz, 2H), 4.28 (s, 3H),
Moreover, predicted constants - partition coefficient
(LogP), molecular polar surface area (TPSA) and molecular
volume (Vol) of synthesized compounds were calculated. If
considered LogP, it can be clearly seen that all substances
1
3
1
1
.33 (s, 9H). C NMR (75 MHz, DMSO d6): ꢀ (ppm)
69.26, 144.84, 124.57, 46.92, 36.10, 29.47.

5
02 Letters in Organic Chemistry, 2009, Vol. 6, No. 6
Jun et al.
Table 1. Structures, In Vitro Biological Activity and Physico-Chemical Parameters of Prepared Monoquaternary Compounds
R
N+
I-
CH3
#
†
‡
±
No
Designation
R
Kdis [mM]
LogP*
TPSA*
Vol*
1
2
3
4
5
6
7
8
9
K024
K025
K001
K143
K144
K146
K020
K017
K147
K016
K148
-CH=NOH
2.254
3.981
0.197
0.902
2.060
0.033
0.622
0.449
0.258
0.169
-1.725
-4.532
-3.252
-3.734
-4.792
-3.13
36.477
46.979
3.885
27.677
62.5
130.958
129.952
99.68
-CONH
-H
2
-CN
116.54
142.247
145.586
187.89
171.089
135.224
165.867
124.5
-C(NH
-N(CH
-CH Phe
2
)=NOH
3
)
2
7.123
3.885
3.885
20.956
3.885
24.113
2
-2.56
-Phe
-2.726
-3.526
-2.815
-4.168
-COCH
3
1
1
0
1
-C(CH
3 3
)
-CH OH
2
No inhibition
#
Dissociation constant characterizing the enzyme-inhibitor complex.
Calculated by software Molinspiration, Bratislava, Slovak Republic.
*
†
Partition coefficients - measure of molecular hydrophobicity.
‡
Molecular polar surface area - sum of surfaces of polar atoms (oxygens, nitrogens and attached hydrogens) in a molecule.
±
Molecular volume - determines transport characteristics of molecules.
are more soluble in water than in octanol. In case of TPSA
sum of surfaces of polar atoms (oxygens, nitrogens and
Thanks to the weak inhibitory potency of prepared
compounds, they could be considered as potential
prophylactics against nerve agents. They differ from
pyridostigmine (currently available nerve agent prophylactic
mean) in the way of AChE inhibition. Pyridostigmine
directly reacts with serine hydroxyl group in the enzyme
active site while these compound bind just thanks to non-
binding interactions, which should be preferred because of
the threat of overdosing by these compounds. If we consider
use of these substances as Alzheimer´s disease treatment,
they fail because of low permeability of BBB. Perhaps their
use as supplement to Alzheimer´s disease treatment should
be examined. They could inhibit peripheral cholinesterases
in blood and other tissues and thanks to this, lower doses of
central acting inhibitors could be used to achieve the same
effect. However, to verify this hypothesis, more in vitro and
in vivo studies should be conducted.
(
attached hydrogens) in a molecule), parameter very useful
for prediction of drug transport properties, the highest value
was reached for compound 2 having carbamoyl group in the
position four. Last parameter - molecular volume -
determines transport characteristics of molecules. The
smallest molecule, the highest probability to penetrate the
barriers, seems to be the non-substituted compound 3.
DISCUSSION
Preparation of new biologically active substances is the
main goal of medical chemists. Cholinesterase inhibitors
seem to be at present time of high interest. These compounds
are used for many purposes - pesticides, Alzheimer´s disease
treatment, etc. [20-25] From the chemical point of view, they
are large group of organic compounds [26, 27]. In this study,
we focused our attention on quaternary monopyridinium
compounds because of their know anticholinesterase activity
and relatively high penetration of the BBB (if compared with
bisquaternary ones) [13,14]. According to the results
obtained, the most strong cholinesterase inhibitor in this
study seems to be compound 6 having dimethylamino group
in the position four at the pyridinium ring.
ACKNOWLEDGEMENT
This work was supported by the Ministry of Defence of
the Czech Republic (KK, DJ), Grant No.FVZ0000604.
REFERENCES
[
1]
Patocka, J.; Kuca, K.; Jun, D. Acta Medica (Hradec Kralove),
004, 47, 215.
Lucic-Vrdoljak, A.; Calic, M.; Radic, B.; Berend, S.; Kuca, K.;
Kovarik, Z. Toxicology, 2006, 228, 41.
Patocka, J.; Jun, D.; Bajgar, J.; Kuca, K. Def. Sci. J., 2006, 56, 775.
Stalc, A.; Sentjurc, M. Biochem. Pharmacol., 1990, 40, 2511.
Due to this fact, three physico-chemical parameters
characterizing BBB penetration were predicted and
compared together with the inhibition potency of tested
compounds.
2
[2]
[
[
3]
4]

Preparation and In Vitro Evaluation of Monoquaternary Inhibitors
Letters in Organic Chemistry, 2009, Vol. 6, No. 6
503
[
[
[
[
[
[
[
[
[
5]
Alkondon, M.; Albuquerque, E.X. J. Pharmacol. Exp. Ther., 1989,
50, 842.
Harris, L.W.; Heyl, W.C.; Stitcher, D.L.; Broomfield, C.A.
Biochem. Pharmacol., 1978, 27, 757.
Kamenicek, J.; Herich, P.; Kuca, K.; Jun, D.; Olsovsky, M. J.
Coord. Chem., 2008, 61, 3525.
Patocka, J.; Cabal, J.; Kuca, K.; Jun, D. J. Appl. Biomed., 2005, 3,
[16]
[17]
Csermely, T.; Petroianu, G.; Kuca, K.; Furesz, J.; Darvas, F.;
Gulyas, Z.; Laufer, R.; Kalasz, H. J. Planar Chromatogr., 2007,
20, 39.
Laufer, R.; Bathori, M.; Csermely, T.; Petroianu, G.; Kuca, K.;
Toth, N.; Kalasz, H. J. Liquid Chromatogr., 2007, 30, 2337.
Giacobini, E., Holmsted, B. Acta Physiol. Scan., 1958, 42, 12.
http://www.molinspiration.com/
Hrabinova, M.; Musilek, K.; Jun, D.; Kuca, K. J. Enzyme Inhib.
Med. Chem., 2006, 21, 515.
Wiesner, J.; Kriz, Z.; Kuca, K.; Jun, D.; Koca, J. J. Enzyme Inhib.
Med. Chem., 2007, 22, 417.
Picha, J.; Kuca, K.; Kivala, M.; Kohout, M.; Cabal, J.; Liska, F. J.
Enzyme Inhib. Med. Chem., 2005, 20, 233.
Patocka, J.; Jun, D.; Kuca, K. Curr. Drug Metab., 2007, 9, 332.
Pohanka, M.; Kuca, K.; Jun, D. Sensor Lett., 2008, 6, 450.
Pohanka, M.; Kuca, K.; Kassa, J. Neuroendocrinol. Lett., 2008, 29,
755.
Kuca, K.; Jun, D.; Musilek, K. Mini Rev. Med. Chem., 2006, 6,
269.
Pohanka, M.; Jun, D.; Kalasz, H.; Kuca, K. Protein Pept. Lett.,
2008, 15, 795.
2
6]
7]
[18]
[19]
[20]
8]
9
1.
9]
Kapkova, P.; Alptuzun, V.; Frey, P.; Erciyas, E.; Holzgrabe, U.
Bioorg. Med. Chem., 2006, 14, 472.
Kapkova, P.; Stiefl, N.; Surig, U.; Engels, B.; Baumann, K.;
Holzgrabe, U. Arch. Pharm. (Weinheim), 2003, 336, 523.
Alptuzun, V.; Kapkova, P.; Baumann, K.; Erciyas, E.; Holzgrabe,
U. J. Pharm. Pharmacol., 2003, 55, 1397.
Bajgar, J.; Fusek, J.; Kuca, K.; Bartosova, L.; Jun, D. Mini Rev.
Med. Chem., 2007, 7, 461.
Sakurada, K.; Matsubara, K.; Shimizu, K.; Shiono, H.; Seto, Y.;
Tsuge, K.; Yoshino, M.; Sakai, I.; Mukoyama, H.; Takatori, T.
Neurochem. Res., 2003, 28, 1401.
[21]
[22]
10]
11]
12]
13]
[23]
[24]
[25]
[26]
[27]
[
[
14]
15]
Lorke, D.E.; Hasan, M.Y.; Nurulain, S.M.; Sheen, R.; Kuca, K.;
Petroianu, G.A. J. Appl. Toxicol., 2007, 27, 482.
Yoon, C.H.; Kim, S.J.; Shin, B.S.; Lee, K.C.; Yoo, S.D. J. Biomol.
Screen., 2006, 11, 13.