Design, synthesis, and biological evaluation of 7H-thiazolo[3,2-b]-1,2,4- triazin-7-one derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.2174/157018010789869343

The docking study on a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives with acetylcholinesterase has been demonstrated. The synthesis and characterization of a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were described. All target compounds were evaluated in vitro for the inhibitory activities against AChE via Ellman colorimetric assay. Most of the target compounds possessed anti-acetylcholinesterase activity. The preliminary structure-activity relationships were discussed.

Full text of DOI:10.2174/157018010789869343

Letters in Drug Design & Discovery, 2010, 7, 5-8
5
Design, Synthesis, and Biological Evaluation of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one
Derivatives as Acetylcholinesterase Inhibitors
Si-Jie Liua, Liu Yanga, Xiao-Guang Liua, Ying Luoa, Zi-Jian Caoa, David Chi Cheong Wanb, Huang-Quan
Linb and Chun Hu*,a
aDepartment of Organic Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016,
China; bDepartment of Biochemistry, The Chinese University of Hong Kong, Hong Kong, SAR, China
Received: April 15, 2009; Revised: August 17, 2009; Accepted: August 20, 2009
Abstract: The docking study on a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives with acetylcholinesterase has been demon-
strated. The synthesis and characterization of a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were described. All target
compounds were evaluated in vitro for the inhibitory activities against AChE via Ellman colorimetric assay. Most of the target com-
pounds possessed anti-acetylcholinesterase activity. The preliminary structure-activity relationships were discussed.
Keywords: Acetylcholinesterase, Inhibitors, Synthesis, Heterocycles, Docking, 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivative.
INTRODUCTION
Docking Studies
Alzheimer’s disease (AD) is a progressive neurodegenerative
disorder, responsible for over 50% of all cases of dementia, which
affects up to 5% of people over 65 years old, while the prevalence
increases to more than 20% of those over 80 years old. While the
disease itself is not fatal, medical complications associated with
AD, usually viral or bacterial infections, lead to the death of the
patient. Thus, AD is the third largest cause of death in the western
world after cardiovascular diseases and cancer [1]. The cholinergic
theory of AD suggests that the selective loss of cholinergic neurons
in AD results in a deficit of acetylcholine in specific regions of the
brain that mediate learning and memory function [2]. Acetylcho-
linesterase (AChE) is a hydrolase that catalyzes the hydrolysis of
neurotransmitter acetylcholine and is present in the most prominent
constituents of central cholinergic pathways. AChE plays a crucial
role in central and peripheral nervous systems [3]. The primary
approach for treating AD has, therefore, focused on increasing the
levels of acetylcholine in the brain by using acetylcholinesterase
inhibitors, such as tacrine, huperzine A, donepezil, galantamine,
rivastigmine, propidium, and ensaculin [4-6].
We carried out docking simulations and molecular dynamics
studies on the AChE-7H-thiazolo[3,2-b]-1,2,4-triazin-7-ones com-
plexes, with the aim to rationalize at molecular level interactions
between the target molecules and AChE and find the better AChE
inhibitors . These dockings also should help to explain the differ-
ences in activity of the compounds tested.
Currently several docking tools are available, such as DOCK,
GOLD, AUTODOCK, Surflex, Glide, FlexX, TPSODOCK, MVD,
HEX, Molsoft ICM-Pro, and 3D-DOCK.
Our strategy was based on the crystallographic structure of the
active-site gorge of Torpedo californica AChE (TcAChE). There-
fore, the crystallographic structure of AChE-Aricept complexes
(1EVE) was selected from Protein Data Bank (PDB). The missing
atoms were properly completed by SPDB Viewer (GSK SPDB
Viewer Version3.7, Swiss), and the structure was prepared by the
structure editing tools DockPrep in UCSF Chimera (UCSF Chi-
mera, Version 1, USA). The AutoDock4 software was used for the
docking study. All water molecules were deleted. This adds a fur-
ther degree of freedom in terms of variability in the docking results.
The minimized structure of the AChE model is shown in Fig. (1),
with AChE represented by an ꢀ-carbon ribbon trace. On the basis
However, the clinical usefulness of marketed AChE inhibitors
is limited mainly by adverse effects on peripheral organs. AD pa-
tients often exhibit psychiatric symptoms, such as irritability, anxi-
ety, and depression [7]. There is still a need to develop more effi-
cient new drugs for AD. So we started a project aimed at the syn-
thesis and the pharmacological study of new acetylcholinesterase
inhibitors. The comprehensive study of the AChE/inhibitor com-
plexes by X-ray crystallography has indicated that AChE possessed
a narrow, long, hydrophobic gorge with three separate ligand bind-
ing sites [8, 9]; a catalysis active site (CAS), the anionic site, and a
peripheral site which is also called peripheral anionic site (PAS).
The active center of AChE contains a catalytic triad (Ser200,
His440, Glu327), which locates at the bottom of the narrow gorge.
Trp84 and Phe330, residues of the anionic site, are located in the
middle of the gorge. Ligands, which either occupied the CAS or the
PAS, could inhibit the AChE activity. In previous research in our
laboratory, a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one
derivatives has been designed and synthesized as acetylcho-
linesterase inhibitors [10]. These target compounds are a kind of
selective ligands, which are designed to allow binding to CAS and
PAS.
*Address correspondence to this author at the Department of Organic Chemistry,
School of Pharmaceutical Engineering, Shenyang Pharmaceutical University,
Shenyang 110016, China; E-mail: chunhu@syphu.edu.cn
Fig. (1). The crystal structure binding mode of 6b in the active site of AChE.
The 6b is rendered as a stick model and is colored yellow.ꢀ
1570-1808/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

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Letters in Drug Design & Discovery, 2010, Vol. 7, No. 1
Liu et al.
of extensive X-ray crystallography and mutagenesis studies, AChE
appears to have a binding domain that interacts with the compound
6b, which includes those residues as Trp84, Gly118, G119, Tyr130,
Ser200, Ala201, Ser286, Phe288, Arg289, Phe290, Phe330,
Tyr334, His440, Gly441, and Tyr442, showed in Fig. (2). In the
active pocket, the compound 6b could form a hydrogen bond with
Tyr130 and hydrophobic interactions with Trp84, Gly117, Gly118,
Tyr121, Ser122, Gly123, Phe290, Phe330, Phe331, Tyr334, His
440, Gly441, and Tyr442, as shown in Fig. (3).
Fig. (2). Docking of compound 6b in the active site of TcAChE model ꢀ
CHEMISTRY
Fig. (3). Interactions of 6b with the active residues of the TcAChE.
The target compounds 5a-5f were obtained in satisfactory
yields, and the synthetic pathways are described in Scheme 1.
In general, compounds 6a-6d were synthesized via a six-step
procedure as described in Scheme and Scheme 2.
4-(Aryl-methylene)-2-methyl-5(4H)-oxazolones (1) were synthe-
sized from aromatic aldehydes and N-acetylglycine. The prepara-
tion of 2 was achieved via 1 using water and acetone. Further
1
The target compound 3-substituted aryl 7H-thiazolo[3,2-b]-1,2,
4-triazin-7-one derivatives (6a-6d) could be obtained with ordinary
Williamson reaction, illustrated in Scheme 2.
O
CH2COOH
NHCOCH3
Ac2O , AcONa
reflux , 7h
acetone , H2O
reflux , 10h
Ar
HCl
Ar
OH
ArCHO
+
O
N
HN
reflux , 7h
O
2
1
yields: 89-93%
yields: 100%
R
O
N
N
N
Ar
O
NH
RCOCH2Cl , AcOH
AcONa , reflux , 8h
NH2NHCSNH2 , K2CO3
reflux , 2h
Ar
Ar
OH
N
H
S
S
O
N
O
5
3
4
yields: 74-83%
yields: 92-93%
yields: 85-89%
Scheme 1. The synthetic route of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives.
OH
OR'
N
N
K2CO3 , acetone
reflux , 24h
N
N
Ar
N
Ar
N
+
R'X
S
O
S
O
6
5
yields: 75-81%
Scheme 2. The synthesis of 3-substituted 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives.

Design, Synthesis, and Biological Evaluation
Letters in Drug Design & Discovery, 2010, Vol. 7, No. 1
7
Table 1. Structures, Yields, Melting Points and of the Target Compounds
No.
Ar
R
Yields, %
mp,°C
5a
5b
5c
5d
5f
4-methoxyphenyl
4-methoxyphenyl
4-methoxyphenyl
phenyl
methyl
4-methylphenyl
82.9
81.5
78.2
79.1
74.8
82.0
77.6
75.6
81.3
179-180
200-202
179-180
224-225
194-196
165-166
165-166
168-169
174-175
4-chlorophenyl
3-methyl-4-hydroxyphenyl
4-methoxyphenyl
phenyl
3-methyl-4-hydroxyphenyl
6a
6b
6c
6d
2-[(2-diethylamino)-2-oxoethoxy]phenyl
{3-methyl-4-[2-(1-piperidinyl)-2-oxoethoxy]}phenyl
4-[(2-diethylamino)-2-oxoethoxy]phenyl
4-[(2-diethylamino)-2-oxoethoxy]phenyl
phenyl
phenyl
4-methoxyphenyl
Table 2. Spectral Data of the Target Compounds
No.
MS(M+H+), IR (cm-1), 1H-NMR and 13C-NMR (ꢀ in ppm)
1
EI-MS: 287(M)+; IR: 3073, 1633, 1567, 1550, 1512, 1483, 1385, 1358, 1300, 1250, 1220, 1180, 1120, 1033, 920; H-NMR (300 MHz, DM-
5a
SO-d6): 7.25 (2H, d, J = 8.7), 7.07 (1H, s), 6.84 (2H, d, J = 8.7), 3.92 (2H, s), 3.71 (3H, s), 2.33 (3H, s).
EI-MS: 363(M)+; IR: 3079, 1654, 1640, 1570, 1539, 1511, 1486, 1384, 1301, 1247, 1178, 1123, 1034; 1H-NMR (600 MHz, DMSO-d6): ꢀ 7.53
(2H, d, J = 7.8), 7.45 (1H, s), 7.24 (2H,d, J = 7.8), 7.20 (2H,d, J = 7.8), 6.86 (2H, d, J = 7.8), 3.83 (2H, s), 3.73 (3H, s); 13C-NMR (600 MHz,
DMSO-d6): ꢀ 164.1, 159.1, 158.2, 154.1, 138.1, 137.4, 128.6, 127.6, 121.6, 116.1, 113.8, 105.5, 54.6, 36.4, 21.3.
5b
EI-MS: 383(M)+; IR: 2926, 1640, 1575, 1513, 1481, 1384, 1302, 1250, 1177, 1115, 1092, 1034; 1H NMR (300 MHz, CDCl3): ꢀ 7.47 (2H, d, J =
8.7), 7.40 (2H, d, J = 8.4), 7.24 (2H,d), 6.84 (3H, d,), 4.06 (2H, s), 3.8 (3H, s).
ESI-MS: 350.0(M+H)+; IR: 3226, 1624, 1604, 1559, 1475, 1405, 1351, 1268, 1199, 1126, 1052;1H-NMR (600 MHz, DMSO-d6): ꢀ 9.83 (1H, s),
7.34 (1H, s), 7.22~7.30 (7H, m), 6.79 (1H, d), 3.99 (2H, s), 2.11 (3H, s); 13C-NMR (600 MHz, DMSO-d6): ꢀ 165.1, 159.4, 158.9, 154.1, 137.8,
136.8, 130.2, 128.6, 127.1, 116.1, 104.5, 37.4, 20.1.
5c
5d
ESI-MS: 380.0 (M+H)+, 780.8 (2M+Na) +; IR: 3235, 2835, 1689, 1618, 1601, 1514, 1469, 1402, 1353, 1302, 1251, 1180, 1126, 1034, 901;
1H-NMR (300 MHz, DMSO-d6): ꢀ 9.78(1H, s), 7.27~7.32 (3H, m), 7.20 (2H, d, J = 8.7Hz), 6.84 (2H, d, J = 8.7Hz), 6.79 (1H, d), 3.90 (2H, s),
3.72 (3H, s), 2.10 (3H, s); 13C-NMR (600 MHz, DMSO-d6): ꢀ 165.2, 159.1, 158.9, 156.1, 153.9, 137.8, 136.8, 131.9, 130.2, 121.3, 116.1, 113.7,
112.9, 106.5, 55.8, 36.9, 21.3.
5f
EI-MS: 448(M)+; IR: 3095, 2978, 2935, 1651, 1579, 1478, 1448, 1383, 1345, 1297, 1251, 1167, 1127, 1067, 954; 1H-NMR (300 MHz, CDCl3):
ꢀ 7.43~7.49 (3H, m), 7.21~7.26 (5H, m ), 7.01~7.07 (2H, m), 4.71(2H, s), 3.87 (2H, s), 3.70 (3H, s), 3.21~3.24 (4H, m), 1.06 (3H, t), 0.98 (3H,
t); 13C-NMR (600 MHz, CDCl3): ꢀ 165.2, 164.8, 159.1, 158.8, 153.9, 135.7, 132.1, 128.6, 127.6, 119.6, 105.7, 66.8, 44.9, 43.6, 35.8, 15.1, 14.2.
ESI-MS:461.0(M+H)+; IR: 3109, 2931, 2785, 1634, 1575, 1478, 1385, 1359, 1305, 1257, 1134, 1037, 957; 1H-NMR (300 MHz, DMSO-d6): ꢀ
7.43 (2H, d), 7.40 (1H, s), 7.36 (1H, s), 7.23~7.30 (5H, m), 6.98 (1H, d), 4.13 (2H, s), 4.0 (2H, s), 2.7 (2H, s), 2.47 (4H, br), 2.13 (3H, m), 1.50
(4H, br), 1.38 (2H, br).
ESI-MS: 449.0(M+H)+; IR: 3092, 2967, 2933, 1648, 1576, 1479, 1383, 1357, 1308, 1253, 1187, 1116, 1071, 1030; 1H-NMR (300 MHz, CDCl3):
ꢀ 7.47 (2H, d, J = 8.7), 7.22~7.35 (5H, m), 6.97 (2H, d, J = 8.7), 6.72 (1H, s), 4.76 (2H, s), 4.12 (2H, s), 3.39~3.48 (4H, m), 1.24~1.29 (3H, t),
1.15~1.20 (3H, t); 13C-NMR (600 MHz, CDCl3): ꢀ 165.9, 164.9, 159.2, 158.6, 154.6, 138.1, 130.8, 130.6, 127.6, 121.1, 115.2, 113.8, 104.6,
67.3, 45.8, 43.2, 36.1, 15.4, 14.6.
6a
6b
6c
ESI-MS: 478.9(M+H)+; IR: 3103, 2974, 2934, 1631, 1581, 1480, 1415, 1384, 1360, 1302, 1249, 1177, 1115, 1032; 1H-NMR (300 MHz, CDCl3):
ꢀ 7.49 (2H, d, J = 8.4), 7.27 (2H, d, J = 9), 7.0 (2H, d, J = 9 ), 6.84 (2H, d, J = 8.4), 6.72 (1H, s), 4.77 (2H, s), 4.05 (2H, s), 3.79 (3H, s),
3.40~3.46 (4H, m), 1.26~1.28 (3H, t), 1.16~1.18 (3H, t); 13C-NMR (600 MHz, CDCl3): ꢀ 165.2, 164.1, 159.4, 158.5, 156.2, 154.4, 136.1, 131.7,
131.4, 130.7, 130.6, 127.6, 120.9, 115.2, 114.7, 113.8, 112.3, 104.3, 66.7, 55.3, 45.6, 42.9, 36.3, 15.6, 14.8.
6d
treatment with 1N hydrochloric acid converted 2 into arylpyruvic
1 and Table 2. All target compounds passed the C, H and N analy-
acids (3). The resulting thiosemicarbazone was cyclized in the
presence of potassium carbonate to generate 4. The condensation of
4 with substituted phenacyl chlorides led to the 6-arylmethyl
-3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives (5a-5f).
ses.
INHIBITION OF AChE
Some
-1,2,4-triazin-7-one derivatives, i.e. 6-phenylmethyl-3-(2-hydroxy
phenyl)-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one, 6-phenylmethyl-
3-(4-hydroxyphenyl)-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one, 6-
other
6-aryl-methyl-3-aryl-7H-thiazolo[3,2-b]
The inhibitory potency against AChE was evaluated by means
of Ellman’s test [13]. Table 3 illustrates the biological activity of
target compounds against freshly prepared human AChE, in com-
parison to the huperzine-A.
phenylmethyl-3-(2-hydroxy-phenyl)-7H-thiazolo[3,2-b]-1,2,4-triazin-7-
one, 6-(4-methoxy-pheylmethyl)-3-(2-hydroxyphenyl)- 7H-thiazolo
[3,2-b]-1,2,4-triazin-7-one, had been synthesized in previous re-
search [10]. Compounds 6a-6d could be obtained by ordinary Wil-
liamson reaction [11, 12]. The structures and spectral characteristics
of the target compounds 5a-5f and 6a-6d were mentioned in Table
In order to check whether aryl ring at C3 position of parent nu-
cleus is necessary for a high inhibitory potency, compound 5a was
additionally studied. In comparison to compound 5a, compounds
5b-5f were observed, respectively, indicating an increase in potency
towards AChE and, along with this, the importance of the phenyl

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Letters in Drug Design & Discovery, 2010, Vol. 7, No. 1
Liu et al.
Table 3. Inhibition of AChE Activities by the targets at 10 μM (n=2)
No.
Inhibition(%)
No.
Inhibition(%)
No.
Inhibition(%)
5a
5b
5c
6.77
50.84
78.08
5d
57.29
59.00
43.94
6b
6c
6d
89.82
72.83
78.87
100
5f
6a
huperzine A (10 μM)
Human AChE (Sigma C-1682) 0.5unit was used. Each performed in double. The incubation time was 20 min, with gentle shake.
piperazine derivatives for acetylcholinesterase inhibition, as a stimulant of
central cholinergic neurotransmission in Alzheimer’s disease. Bioorg. Med.
Chem. Lett., 2006, 16, 3932-3936.
José, M.C.; Rafael, L.; Cristóbal de los, R.; Antonio, G.; José, T.; Manuela,
G.; Mercedes, V. Novel multipotent tacrine-dihydropyridine hybrids with
improved acetylcholinesterase inhibitory and neuroprotective activities as
potential drugs for the treatment of Alzheimer's disease. J. Med. Chem.,
2006, 49(26), 7607-7610.
Tang, H.; Ning, F.X.; Wei, Y.B.; Huang, S.L.; Huang, Z.S. Derivatives of
oxoisoaporphine alkaloids: A novel class of selective acetylcholinesterase
inhibitors. Bioorg. Med. Chem. Lett., 2007, 17(13), 3765-3768.
Lahiri, D.K.; Rogers, J.T.; Greig, N.H.; Sambamurti, K. Rationale for the
development of cholinesterase inhibitors as anti-Alzheimer agents. Curr.
Pharm. Des., 2004, 10(25), 3111-3119.
Shen, Q.; Peng, Q.; Shao, J.L.; Liu, X.F.; Huang, Z.S.; Pu, X.Z.; Ma, L.
Synthesis and biological evaluation of functionalized coumarins as acetyl-
cholinesterase inhibitors. Eur. J. Med. Chem., 2005, 40(12), 1307-1315.
Wen, H.; Zhou, Y.Y.; Lin, C.L.; Ge, H.; Ma L.; Wang, Z.H. Methyl
2-(2-(4-formylphenoxy)acetamido)-2-substituted acetate derivatives: A new
class of acetylcholinesterase inhibitors. Bioorg. Med. Chem. Lett., 2007,
17(8), 2123-2125.
Kogen, H.; Toda, N.; Tago, K.; Marumoto, S.; Takami, K.; Mayuko, O.;
Naho, Y.; Kazuo, K. Design and synthesis of dual inhibitors of acetylcho-
linesterase and serotonin transporter targeting potential agents for Alzhei-
mer’s disease. Org. Lett., 2002, 4(20), 3359-3362.
Carlos, H.T.P.; Silva, D.; Campo, V.L.; Carvalho, I.; Taft, A. Molecular
modeling, docking and ADMET studies applied to the design of a novel hy-
brid for treatment of Alzheimer’s disease. J. Mol. Graphics Modell., 2006,
25(2), 169-175.
Kapková, P.; Stiefl, N.; Sürig, U. Synthesis, biological activity, and docking
studies of new acetylcholinesterase inhibitors of the bispyridinium type.
Arch. Pharm. Pharm. Med. Chem., 2003, 336, 523-540.
Liu, S.J.; Yang, L.; Jin, Z.; Huang, E.F.; Wan, D.C.C.; Lin, H.Q. Design,
synthesis, and biological evaluation of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one
derivatives as novel acetylcholinesterase inhibitors. ARKIVOC, 2009, (x),
333-348.
rings. The compound 5c, which possessed electron withdrawing
groups, such as chloro at the para position on the phenyl ring,
showed higher AChE inhibitory activity. Compound 5b with
weaker electron donating group, the methyl group at the para posi-
tion on the phenyl ring, showed a decreasing inhibitory activity,
which suggested that electron withdrawing group in these com-
pounds could help them to access to the enzyme-binding site. The
compounds 5d-5f showed moderate activity, this was probably due
to the presence of hydroxy group, which would be able to form
hydrogen bond with AChE receptor. The modification based on the
structure of compounds 5 was a practical approach to increase its
AChE inhibitory activities. While the compound 6a with small
substituent at the ortho position on the phenyl ring led to a huge
decrease in activity than that bearing small substituent at the para
position on the phenyl ring (compounds 6c and 6d), indicating the
steric hindrance could limit the access of compounds to the en-
zyme-binding site. Compound 6b possessed two substituent groups
at the meta and para positions on the phenyl ring exhibited an in-
crease in AChE inhibitory effect compared to those bearing one
group at the para position.
[2]
[3]
[4]
[5]
[6]
[7]
[8]
These phenomena implied that both the steric and electronic
effects might play a significant role in influencing the activity.
CONCLUSION
In conclusion, 6-arylmethyl-3-aryl-7H-thiazolo[3,2-b]-1,2,4-
triazin-7-one derivatives are a class of active and selective AChE
inhibitors. Further efforts aiming at developing potent AChE in-
hibitors based on modification of compounds 5 and 6 would be
continued in our laboratory.
[9]
[10]
ACKNOWLEDGMENT
[11]
Roshan, A.; Aurangzeb, H.; Saleh, A. Synthesis antimicrobial activity of
6-Benzyl/p-substituted-benzyl-1,2,4-triazine-3,5-(2H,4H)-dithione. Indin J.
Chem., 1987, 26B(4), 393.
Holla B. S.; Malini K. V.; Sarojini B. K. A novel three-component synthesis
of triazinothiazolones. Synth. Commun., 2005, 35(3), 333-340.
Ellman, G.L.; Courtiney, K.D.; Andes, V. Anew and rapid colorimetric
determination of acethylcholinesterase activity. Biochem. Pharmaco., 1961,
7(2), 88-95.
This work was partially supported by the National Science
Foundation of China (NFSC) for the grant No. 40724053. The
authors would like to thank Ms. He Zhu for measuring the NMR
spectra.
[12]
[13]
REFERENCES
[1]
Sadashiva, C.T.; Narendra, J.N.; Sharath, C.; Ponnappa, K.C.; Veerabasappa,
T.; Rangappaa, S. Synthesis and efficacy of 1-[bis(4-fluorophenyl)-methyl]