Synthesis and biological evaluation of 7H-thiazolo[3,2-b]-1,2,4-triazin-7- one derivatives as dual binding site acetylcholinesterase inhibitors

Article abstract of DOI:10.1016/j.ejmech.2014.05.020

A series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives 7a-i were synthesized and evaluated as novel acetylcholinesterase (AChE) inhibitors. All target compounds were evaluated in vitro for the inhibitory activities against AChE via Ellman colorimetric assay. Compound 7c showed an excellent (89.82%) inhibitory activity. The molecular docking studies revealed that 7c, 7d and 7g, with the lateral chain in the para position of the phenyl ring, possessed an optimal docking pose and can perfectly fit into the catalytic active site (CAS) and peripheral anionic site (PAS), simultaneously, and, consequently, exhibited higher inhibitory potency than 7b that bears the same lateral chain as 7g, but in the ortho position of the phenyl ring.

Full text of DOI:10.1016/j.ejmech.2014.05.020

Accepted Manuscript
Synthesis and biological evaluation of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one
derivatives as dual binding site acetylcholinesterase inhibitors
Sijie Liu, Ruofeng Shang, lanxiang shi, David Chi-Cheong Wan, Huangquan Lin
PII:
S0223-5234(14)00434-6
10.1016/j.ejmech.2014.05.020
EJMECH 6976
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 August 2013
Revised Date: 19 November 2013
Accepted Date: 5 May 2014
Please cite this article as: S. Liu, R. Shang, lanxiang shi, D. Chi-Cheong Wan, H. Lin, Synthesis
and biological evaluation of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives as dual binding site
acetylcholinesterase inhibitors, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.05.020.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis and biological evaluation of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one
derivatives as dual binding site acetylcholinesterase inhibitors
Sijie Liua, Ruofeng Shangb*, lanxiang shia, David Chi-Cheong Wanc, Huangquan Linc
aCollege of Chemical Engineering, Shijiazhuang University, Shijiazhuang 050035,China
bKey Laboratory of New Animal Drug Project of Gansu Province; Key Laboratory of Veterinary Pharmaceutical Development,
Ministry of Agriculture, P. R. China; Lanzhou Institute of Animal Science and Veterinary Pharmaceutics Science, Chinese Academy of
Agricultural Sciences, Lanzhou, 730050, China
cDepartment of Biochemistry, The Chinese University of Hong Kong, Hong Kong SAR, China
Corresponding author: Tel.: +86 931 2115286; fax: +86 931 2115951. E-mail address: shangrf1974@163.com
(Ruofeng Shang).
Author: Tel. T +86 311 66617323. E-mail address: liusijie1982@163.com (Sijie Liu).
Address: College of Chemical Engineering, Shijiazhuang University, No. 6, New Technology Development
District, Shijiazhuang, 050035, P.R. China.
Abstract:
A series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives 7a-i were synthesized and evaluated as novel
acetylcholinesterase (AChE) inhibitors. All target compounds were evaluated in vitro for the inhibitory activities
against AChE via Ellman colorimetric assay. Compound 7c showed an excellent (89.82%) inhibitory activity. The
molecular docking studies revealed that 7c, 7d and 7g, with the lateral chain in the para position of the phenyl ring,
possessed an optimal docking pose and can perfectly fit into the catalytic active site (CAS) and peripheral anionic
site (PAS), simultaneously, and, consequently, exhibited higher inhibitory potency than 7b that bears the same
lateral chain as 7g, but in the ortho position of the phenyl ring.
Keywords: acetylcholinesterase; synthesis; inhibitors; docking; Alzheimer’s disease
1. Introduction
Alzheimer’s disease (AD) is a complex neurodegenerative disorder that attacks the central nervous system
through progressive degeneration of its neurons. Patients with AD develop dementia which becomes more severe
when the disease progresses. The cholinergic hypothesis states that the cognitive decline in AD is secondary to
deficits in central cholinergic neurotransmission resulting from a loss of acetylcholine. Acetylcholinesterase (AChE)
inhibitors enhance central cholinergic function by inhibiting the enzyme to degrade acetylcholine, thereby
increasing the availability of acetylcholine to stimulate nicotinic and muscarinic receptors within the brain. Since
their introduction into clinical practice, AChE inhibitors have been, and remained, the standard approach to the
symptomatic treatment of AD [1]. A number of AChE inhibitors had been reported, such as tacrine, galanthamine,
huperzine-A, donepezil and rivastigmine (Fig. 1) [2-5].
Solution of the 3D structure of Torpedo californica acetylcholinesterase in 1991 opened up new horizons in
research on an enzyme that had already been the subject of intensive investigation. The crystal structure of human
AChE had been reported in 2000 [6]. One of the striking structural features of the AChE revealed from the X-ray
analysis is the presence of a narrow, long, hydrophobic gorge which is approximately 20Å deep [7]. At the bottom
of the gorge a catalytic triad (Ser203, His447 and Glu334) lies in catalytic active site (CAS). The second binding is
1

ACCEPTED MANUSCRIPT
referred to as the peripheral anionic site (PAS), including Tyr72, Asp74, Tyr124, Glu285, Trp286 and Tyr341, and
is known to be located at the gorge mouth around 18Å away from the active site. Furthermore, in the middle of the
gorge, Trp86, Tyr133, Tyr337 and Tyr449 form an anionic binding site, a binding site for the quaternary nitrogen
of their substrates and some ligands [8] (Fig. 2).
Considering the non-cholinergic aspects of the cholinergic enzyme, AChE inhibitors which can interact with
both the CAS and PAS may simultaneously alleviate the cognitive deficit in Alzheimer’s patients and what it is
more important, avoid the assembly of β-amyloid peptide which represents a new way to delay the
neurodegenerative process [9].
In our previous work, 3-aryl-6-arylmethyl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were designed and
synthesized. Some compounds exhibited inhibitory activity against AChE, and interacted with the PAS and the
CAS of AChE [10-12]. In continuation of our research in this field,
a
novel series of
7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were synthesized to serve as highly effective inhibitors of AChE.
In addition, the classic Ellman test [13] was performed to assess the AChE inhibitory activities of the synthesized
compounds.
2. Results and Discussion
2.1. Chemistry
The synthetic pathways of the 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were outlined in Scheme 1. At
the first stage, aromatic aldehydes were cyclized with N-acetylglycine in acetic anhydride, which led to a high yield
of 4-(arylmethylene)-2-methyl-5(4H)-oxazolones 2. Then, the obtained 2 was treated with acetone and water to
obtain 2-acetamido-3-aryl-2-propenoic acid 3. The compound 3 was further hydrolyzed with 1 N hydrochloric acid
to provide aryl pyruvic acids 4. Compound 4 was condensed with thiosemicarbazone to produce
6-(arylmethyl)-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-ones 5 by using K2CO3 in 50% ethanol . The cyclization
of 4 with different substituted phenacyl chlorides in NaOAc/HOAc provided 6 in 68–80% yields. Finally, the target
products 7 were gained by the Williamson reaction of 6 with different substituted alkyl chloride in K2CO3/acetone.
2.2. Inhibition of AChE
Inhibitory activities of target compounds on AChE were determined in comparison to the huperzine-A and the
results data were reported in Table 1. All compounds showed good activities for AChE inhibition. Among all the 9
derivatives examined, three compounds 7c, 7d and 7g showed excellent inhibitory activity in the range from 73.92
- 89.82 %, while the 7e and 7f showed moderate inhibitory activity against AchE. Numerous changes of the
substituents in the aryl ring at C3 position of parent nucleus with 2-(1-piperidinyl) ethoxy,
2-(dimethylamino)-2-oxoethoxy groups succeed in increasing inhibitory activities against AChE. For example,
compound 7c exhibited the best inhibitory activity to AChE with the mean inhibition value of 89.82%. However,
all the synthesized compounds showed lower inhibitory activities than the reference drug huperzine-A. The
compounds with the substituents at the ortho position on the aryl ring at C3 position of parent nucleus led to a huge
decrease in activity than that bearing the substituents at the para position. For example compound 7g
showed stronger inhibitory activity than compound 7b. According to biological evaluation results, the introduction
of methyl groups on the benzene ring increases inhibitory potency because both 7c and 7d are more effective than
7b.
2.3. Docking studies
Previous recombinant human AChE structures (1B41) of the catalytic core have been solved to date, of which
2

ACCEPTED MANUSCRIPT
none are in complex with therapeutic drugs used for the treatment of disease. There is also a structure of
recombinant human AChE (4EY7) in complex with donepezil. The comparison of binding site amino acid 3D
structures revealed an overall identity, only conformations of Tyr337 are small different (Fig. 3). So docking
simulations were performed with the recombinant human AChE (PDB ID: 4EY7) [14].
To investigate the binding mode of the series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives, docking
simulations were performed with Autodock 4 software. The docking simulations reveal all target compounds
exhibited multiple binding modes with 4EY7 and the binding free energies (ꢀGb) of compounds 7a-i being the
-10.46, -11.15, -12.54, -12.49, -12.37, -12.29, -12.45, -10.89 and -10.57 kcal/mol, respectively. The calculations
with the flexible docking protocol placed the 9 compounds correctly into the binding pocket and the docking results
revealed some different binding patterns as presented in Fig. 4, which shows a superposition of the 9 docked
compounds. The docking results revealed all the compounds could suitably interact with the CAS and part of
compounds interact with the PAS of the enzyme (Fig. 4.). For example, 7b, 7c, 7d and 7g formed a hydrogen bond
with Tyr124, while 7c, 7d and 7g formed π-π interaction with Trp286. In the middle of the gorge, the morpholine
moiety of 7a, piperidinyl of 7c, 7d and 7d forming protonation state interacted with Trp86 by means of cation-π
interaction (Fig. 5). Protonation state was formed while Nitrogen-containing substituents on the aryl ring at C3
position of parent nucleus, such as 7c, 7d and 7f.
The analysis of the docking results, revealed the compounds 7c, 7d and 7g possessing lower binding free
energies and optimal docking pose. Also compounds 7c, 7d and 7g were all able to protrude out of the AChE gorge,
a feature which is considered important for the AChE-induced Aβ aggregation inhibition. The common feature of
these compounds is that an aminoalkyl side chain in the para position of the phenyl ring. While compound 7b, with
the same lateral chain as 7g but in the ortho position of the phenyl ring, showed the higher binding free energies
and only two hydrogen bonds.
Comparing the docking results and the inhibitory activities of target compounds, there is a reasonable
correspondence between theoretical predictions and experimental data, which indicating compounds with the lateral
chain in the para position of the phenyl ring, possessed an optimal docking pose and can perfectly fit into the
catalytic active site (CAS) and peripheral anionic site (PAS).
3. Conclusion
In conclusion, 6-arylmethyl-3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were synthesized here and
exhibited excellent inhibitory activities against AChE. Among them, the compound 7c which have 2-(1-piperidinyl)
ethoxy in the aryl ring at C3 position exerted more potent inhibitory activity than other compounds, with 89.82%
inhibition. Molecular docking results revealed information about the binding mode between inhibitors and human
AChE, illustrating that 7c, 7d and 7g might behave as dual binding site inhibitors, while the compounds bearing
para substituents on the benzene ring increased binding affinities.
4. Experimental Section
4.1. Chemistry
Reagents and solvents were purchased from common commercial suppliers and were used without further
purification. All melting points were taken in open capillary tubes and are uncorrected. Nuclear magnetic resonance
spectra were recorded in CDCl3 or DMSO solutions, using Bruker 300 MHz spectrometers or 600 MHz
spectrometers. The mass spectra (MS) were obtained by electronic impact (EI) at 70eV in an Agilent spectrometer
(with direct insertion probe) or by electrospray (ESI) in a Waters spectrometer. The IR spectra were obtained using
3

ACCEPTED MANUSCRIPT
a Perkin-Elmer 298 spectrometer (Perkin-Elmer, Norwalk, CT, USA). The C, H and N analyses were performed on
a Perkin Elmer 240C elemental analyzer.
4.2. General procedure for synthesis of 4-(Arylmethylene)-2-methyl-5(4H)-oxazolones (2)
A mixture of aromatic aldehyde (100 mmol), N-acylglycine (10.7 g, 120 mmol), sodium acetate (51 g, 500 mmol)
were mixed and heated at 120°C for 7h. After completion of the reaction, the resulting solution was kept in
refrigerator overnight to obtain a solidified mass which was triturated with cold water and ethanol. The resulting
yellow crystals were collect and dried to give 4-(arylmethylene)-2-methyl-5(4H)-oxazolone (2).
4.2.1. 4-(Phenylmethylene)-2-methyl-5(4H)-oxazolone (2a)
This compound was obtained as a yellow solid, 100% yield; mp: 149-151°C; ESI-MS (m/z): 188.1 (M+H+),
398.1 (2M+Na+).
4.2.2.4-[(4-Methoxyphenyl)methylene]-2-methyl-5(4H)-oxazolone (2b)
This compound was obtained as a yellow solid, 100% yield; mp: 156-158°C; ESI-MS (m/z): 218.1 (M+H+),
457.0 (2M+Na+).
4.3. General procedure for synthesis of 2-Acetamido-3-aryl-2-propenoic acids (3)
4-(Arylmethylene)-2-methyl-5(4H)-oxazolone (10.85 g, 50 mmol), acetone (85ml), and H2O (70 mL) were
refluxed for 10h. The solvent was evaporated to give 2-acetamido-3-arylacrylic acids as a residual solid.
4.3.1. 2-Acetamido-3-phenyl-2-propenoic acid (3a)
This compound was obtained as a yellow solid, 89% yield; mp: 193-194°C; ESI-MS (m/z): 206.0(M+H+), 433.3
(2M+Na+).
4.3.2. 2-Acetamido-3-(4-methoxyphenyl)-2-propenoic acid (3b)
This compound was obtained as a yellow solid, 93% yield; mp: 219-221°C; ESI-MS (m/z): 236.1(M+H+), 493.2
(2M+Na+).
4.4. General procedure for synthesis of Arylpyruvic acids (4)
Compound 3 (10 mmol) and 1mol/L HCl aqueous solution (60 mL) were refluxed for 7 h. After that the resulting
mixture was allowed to cool to room temperature. And the resulting precipitate was collected by filtration. The
filtrate was extract with ethyl acetate (3×20 mL). The organic extract was evaporated to give a residue which was
combined with the filtered precipitate. The total product was dried under vacuum and was recrystallized form
ethanol.
4.4.1. Phenylpyruvic acid (4a)
This compound was obtained as a yellow solid, 89% yield; mp: 153-154°C; ESI-MS (m/z): 351.2 (2M+Na+).
4.4.2. 4-Methoxyphenylpyuvic acid (4b)
This compound was obtained as a yellow solid, 90% yield; mp: 184-185°C; ESI-MS (m/z): 194.9 (M+H+), 411.1
(2M+Na+).
4.5. General procedure for synthesis of 6-(Arylmethyl)-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-ones (4)
A mixture of 4-arylpyruvic acid (10 mmol), thiosemicarbazone (10 mmol), potassium carbonate, ethanol (25 mL)
and water (25 mL) were refluxed for 2 h, cooled to room temperature and treated with HCl until effervescence to
go to completion, the solid formed was collected and crystallized from ethanol.
4.5.1. 6-(Phenylmethyl)-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one (5a)
This compound was obtained as a white solid, 93% yield; mp: 185-187°C; ESI-MS (m/z): 219.9 (M+H+), 461.1
4

ACCEPTED MANUSCRIPT
(2M+Na+).
4.5.2. 6-[(4-Methoxyphenyl)methyl]-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one (5b)
This compound was obtained as a white solid, 92% yield; mp: 166-167°C; ESI-MS (m/z): 249.8 (M+H+).
4.6. General procedure for synthesis of 6-Arylmethyl-3-hydroxylphenyl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-ones (6)
A mixture of 6-arylmethyl-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one (10 mmol), substituted phenacyl
chloride (10 mmol) and AcONa/AcOH (2 g/20 mL) were refluxed for 8-24 h. After completion of the reaction as
indicated by TLC, the reaction liquid was cooled to room temperature, which crystallized to afford
6-arylmethyl-3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one.
4.6.1. 6-Benzyl-3-(2-hydroxyphenyl)thiazolo[3,2-b]-1,2,4-triazin-7-one (6a)
This compound was obtained as a white crystal, 85.5% yield; mp: 227-230 °C; 1H-NMR (600 MHz, DMSO): δ
10.00 (1H, s), 7.38 (1H, s), 7.18~7.35 (7H, m), 6.99 (1H, d, J = 7.8Hz), 6.88 (1H, t, J = 7.2Hz), 3.88 (2H, s); MS
(m/z): 51, 65, 77, 90, 103, 121, 132, 149, 150, 168, 185, 218, 220, 335; IR (KBr): υ 3219, 1697, 1646, 1618, 1482,
1384, 1350, 1296, 1258, 1240, 1181, 1157, 1108, 1073, 1028, 929, 861, 838, 752, 703, 666, 611 cm-1.
4.6.2. 6-(4-Methoxybenzyl)-3-(2-hydroxyphenyl)thiazolo[3,2-b]-1,2,4-triazin-7-one (6b)
This compound was obtained as a white crystal, 81.2% yield; mp: 218-219 °C; 1H-NMR (300 MHz, DMSO): δ
9.99 (1H, s), 7.37 (1H, s), 7.32~7.58 (2H, m), 7.18 (2H, d, J = 8.7Hz), 6.99 (1H, d, J =8.7 Hz), 6.89 (1H, t, J =
7.5Hz), 6.81 (2H, d, J = 8.7Hz), 3.80 (2H, s), 3.72 (3H, s); MS (m/z): 51, 63, 77, 89, 104, 121, 132, 147, 159, 174,
192, 193, 218, 231, 282, 350, 365; IR (KBr): υ 3102, 2965, 1663, 1621, 1571, 1513, 1486, 1384, 1294, 1267, 1243,
1180, 1127, 1107, 1047, 1025, 943, 921, 879, 842, 803, 773, 682, 649, 610 cm-1.
4.6.3. 6-Benzyl-3-(3-methyl-4-hydroxyphenyl)thiazolo[3,2-b]-1,2,4-triazin-7-one (6c)
This compound was obtained as a white crystal, 79.1% yield; mp: 224-225 °C; 1H-NMR (600 MHz, DMSO): δ
9.83 (1H, s), 7.34 (1H, s), 7.22~7.30 (7H, m), 6.79 (1H, d, J =8.4 Hz), 3.99 (2H, s), 2.11 (3H, s); ESI-MS (m/z):
350.0 (M+H+), 720.9 (2M+Na+); IR (KBr): υ 3226, 1624, 1604, 1559, 1475, 1405, 1351, 1268, 1199, 1126, 1052,
897, 867, 819, 738, 702, 617 cm-1.
4.6.4. 6-(4-Methoxybenzyl)-3-(3-methyl-4-hydroxyphenyl)thiazolo[3,2-b]-1,2,4-triazin-7-one (6d)
This compound was obtained as a white crystal, 78.3% yield; mp: 194-196 °C; 1H-NMR (300 MHz, DMSO): δ
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, J = 8.4Hz), 3.90 (2H,
s), 3.72 (3H, s), 2.10 (3H, s); ESI-MS (m/z): 380.0 (M+H+), 780.8 (2M+Na+); IR (KBr): υ 3235, 2835, 1689, 1618,
1601, 1514, 1469, 1402, 1353, 1302, 1251, 1180, 1126, 1034, 901, 814, 765, 668, 611 cm-1.
4.6.5. 6-Benzyl-3-(4-hydroxyphenyl)thiazolo[3,2-b]-1,2,4-triazin-7-one (6e)
This compound was obtained as a white crystal, 79.2% yield; mp: 245-247 °C; 1H-NMR (600 MHz, DMSO): δ
9.92 (1H, s), 7.46 (2H, d, J = 8.4Hz), 7.33 (1H, s), 7.23~7.30 (5H, m), 6.78 (2H, d, J = 8.4Hz), 3.98 (2H, s);
ESI-MS (m/z): 335.9 (M+H+); IR (KBr): υ 3230, 1620, 1604, 1558, 1511, 1472, 1441, 1385, 1346, 1299, 1275,
1230, 1206, 1177, 1127, 1050, 842, 776, 757, 749, 722, 702, 655, 628 cm-1.
4.6.6. 6-(4-Methoxybenzyl)-3-(4-hydroxyphenyl)thiazolo[3,2-b]-1,2,4-triazin-7-one (6f)
This compound was obtained as a white crystal, 80.1% yield; mp: 188-189 °C; 1H-NMR (600 MHz, DMSO): δ
10.0 (1H, s), 7.48 (2H, d, J = 8.4Hz), 7.33 (1H, s), 7.21 (2H, d, J = 8.4Hz), 6.87 (2H, d, J = 8.4Hz), 6.80 (2H, d, J =
8.4Hz), 3.91 (2H, s), 3.76 (3H, s); ESI-MS (m/z): 366.0 (M+H+), 752.8 (2M+Na+); IR (KBr): υ 3119, 1719, 1610,
1511, 1476, 1385, 1279, 1247, 1176, 1123, 1029, 838, 752, 668 cm-1.
4.7. General procedure for synthesis of 6-Arylmethyl-3-aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-ones (7)
5

ACCEPTED MANUSCRIPT
A mixture of 6-arylmethyl-3-hydroxylphenyl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (10 mmol), substituted alkyl
chlorides (10 mmol), potassium carbonate (50 mmol), KI (1 mmol) and acetone (25 mL) was refluxed for 24h. The
solvent was evaporated, a residual solid was given and recrystallized form ethanol.
4.7.1. 6-benzyl-3-(2-(2-morpholinoethoxy)phenyl)-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (7a)
This compound was obtained as a white crystal, 39.0% yield; mp: 145-147 °C; 1H-NMR (600 MHz, DMSO-d6): δ
7.50 (1H, t, J = 8.7Hz), 7.42 (2H, d, J = 8.7Hz), 7.22 (5H, m), 7.13 (1H, d, J = 8.7Hz), 7.05 (1H, t, J = 8.7Hz), 4.00
(2H, t, J = 5.2Hz), 3.89 (2H, s), 3.40 (4H, t, J = 4.2Hz), 2.36 (2H, t, J = 5.2Hz), 2.18 (4H, t, J = 4.2Hz); EI-MS
(m/z): 56, 70, 91, 100, 111, 134, 149, 163, 192, 218, 330, 448(M)+; IR (KBr): υ 3083, 2851, 2364, 1636, 1478,
1384, 1289, 1264, 1116, 1042, 961, 857, 756, 701 cm-1; Anal. Calcd For C24H24N4O3S: C, 64.27; H, 5.39; N,12.49.
Found: C, 64.35; H, 5.35; N, 12.42.
4.7.2. 6-(4-Methoxybenzyl)-3-{2-[2-(dimethylamino)-2-oxoethoxy]phenyl}thiazolo[3,2-b]-1,2,4-triazin-7-one (7b)
This compound was obtained as a white crystal, 81.5% yield; mp: 238-239 °C; 1H-NMR (600 MHz, DMSO-d6):
δ 7.34~7.48 (3H, m), 7.12 (2H, d, J = 8.4Hz), 7.05 (2H, m), 6.79 (2H, d, J = 8.4Hz), 4.77 (2H, s), 3.80 (2H, s), 3.70
(3H, s), 2.89 (3H, s), 2.78 (3H, s); ESI-MS (m/z): 451.0 (M+H+), 472.9 (M+Na+); IR (KBr): υ 3123, 2928, 1666,
1642, 1590, 1567, 1511, 1478, 1418, 1383, 1356, 1300, 1238, 1179, 1122, 1064, 1045, 1028, 921, 849, 811, 767,
736 cm-1; Anal. Calcd For C23H22N4O4S: C, 61.32; H, 4.92; N,12.44. Found: C, 61.26; H, 4.97; N, 12.48.
4.7.3. 6-Benzyl-3-{3-methyl-4-[2-(1-piperidinyl)ethoxy]phenyl}-thiazolo[3,2-b]-1,2,4-triazin-7-one (7c)
This compound was obtained as a white crystal, 43.3% yield; mp: 168-169℃; 1H-NMR (600 MHz, DMSO-d6): δ
7.43 (1H, d, J = 7.8Hz), 7.39 (H, s), 7.36 (H, s), 7.23~7.30 (5H, m), 6.98 (1H, d, J = 8.4Hz), 4.13 (2H, s), 3.88 (2H,
s), 2.70 (2H, s), 2.46 (4H, s), 2.13 (3H, s), 1.50 (4H, br), 1.39 (2H, br); ESI-MS (m/z): 461.0 (M+H)+; IR (KBr): υ
3109, 2931, 2785, 1634, 1575, 1478, 1385, 1359, 1305, 1257, 1134, 1037, 957, 883, 859, 815, 799, 767, 750, 708
cm-1; Anal. Calcd For C26H28N4O2S: C, 67.80; H, 6.13; N,12.16. Found: C, 67.74; H, 6.17; N, 12.19.
4.7.4. 6-(4-Methoxybenzyl)-3-{3-methyl-4-[2-(1-piperidinyl)ethoxy]phenyl}thiazolo[3,2-b]-1,2,4-triazin-7-one (7d)
1
This compound was obtained as a white crystal, 82.0% yield; mp: 165-166 °C; H-NMR (600 MHz, CDCl3): δ
7.36 (1H, d, J = 9.0Hz), 7.32 (1H, s), 7.29 (2H, d, J = 8.4Hz), 6.84 (1H, d, J = 7.8Hz), 6.83 (2H, d, J = 8.4Hz), 6.68
(1H, s), 4.21 (2H, br), 4.06 (2H, s), 3.80 (3H, s), 2.90 (2H, br), 2.59 (4H, br), 2.24 (3H, s), 1.65 (4H, br), 1.48 (2H,
br); ESI-MS (m/z): 491.0 (M+H+), 981.0 (2M+H+); IR (KBr): υ 3067, 2933, 2852, 1632, 1565, 1513, 1486, 1384,
1356, 1302, 1248, 1177, 1135, 1034, 952, 863, 802, 764 cm-1; Anal. Calcd For C27H30N4O3S: C, 66.10; H, 6.16;
N,11.42. Found: C, 66.16; H, 6.12; N, 11.48.
4.7.5. 6-Benzyl-3-{4-[2-(dimethylamino)-2-oxoethoxy]phenyl}thiazolo[3,2-b]-1,2,4-triazin-7-one(7e)
This compound was obtained as a white crystal, 78.2% yield; mp: 144-146°C; ESI-MS (m/z): 421.0 (M+H)+;
1H-NMR (300 MHz, CDCl3): δ 7.47 (2H, d, J = 8.7Hz), 7.25~7.36 (5H, m), 6.98 (2H, d, J = 8.7Hz), 6.72 (1H, s),
4.77 (2H, s), 4.12 (2H, s), 3.13 (3H, s), 3.02 (3H, s) ; ESI-MS (m/z): 421.0 (M+H+) ; IR (KBr): υ 3077, 2928, 1636,
1576, 1507, 1484, 1417, 1354, 1307, 1255, 1185, 1122, 1069, 827, 755, 704 cm-1; Anal. Calcd For C22H20N4O3S: C,
62.84; H, 4.79; N,13.32. Found: C, 62.74; H, 4.82; N, 13.36.
4.7.6. 6-Benzyl-3-{4-[2-(1-piperidinyl)ethoxy]phenyl}thiazolo[3,2-b]-1,2,4-triazin-7-one (7f)
1
This compound was obtained as a white crystal, 80.5% yield; mp: 185-187 °C; H-NMR (300 MHz, CDCl3): δ
7.46 (2H, d, J = 8.8Hz), 7.25~7.36 (5H, m), 6.92 (2H, d, J = 8.8Hz), 6.72 (1H, s), 4.18 (2H, t, J = 6.0Hz), 4.12 (2H,
s), 2.83 (2H, t, J = 6.0Hz), 2.56 (4H, br), 1.65 (4H, m), 1.48 (2H, m); ESI-MS (m/z): 447.0 (M), 893.0 (2M), 915.9
(M+Na+); IR (KBr): υ 3109, 2931, 2851, 1634, 1579, 1507, 1480, 1416, 1386, 1356, 1293, 1253, 1173, 1036, 933,
824, 767 cm-1; Anal. Calcd For C25H26N4O2S: C, 67.24; H, 5.87; N,12.55. Found: C, 67.34; H, 5.82; N, 12.51.
6

ACCEPTED MANUSCRIPT
4.7.7. 6-(4-Methoxybenzyl)-3-{4-[2-(dimethylamino)-2-oxoethoxy]phenyl}thiazolo[3,2-b]-1,2,4-triazin-7-one (7g)
1
This compound was obtained as a white crystal, 76.5% yield; mp: 173-175°C; H-NMR (300 MHz, CDCl3): δ
7.49 (2H, d, J = 8.6Hz), 7.27 (2H, d, J = 8.4Hz), 7.00 (2H, d, J = 8.6Hz), 6.84 (2H, d, J = 8.4Hz), 6.71 (1H, s), 4.78
(2H, s), 4.05 (2H, s), 3.79 (3H, s), 3.13 (3H, s), 3.02 (3H, s) ; ESI-MS (m/z): 451.1 (M+H+); IR (KBr): υ 3072,
2933, 1632, 1513, 1482, 1417, 1357, 1301, 1249, 1118, 1068, 1027, 816, 764 cm-1; Anal. Calcd For C23H22N4O4S:
C, 61.32; H, 4.92; N,12.44. Found: C, 61.37; H, 4.86; N, 12.50.
4.7.8.6-(4-Methoxybenzyl)-3-{4-[2-(diethylamino)ethoxy]phenyl}thiazolo[3,2-b]-1,2,4-triazin-7-one (7h)
1
This compound was obtained as a white crystal, 80.5% yield; mp: 157-159 °C; H-NMR (300 MHz, CDCl3): δ
7.47 (2H, d, J = 8.7), 7.26 (2H, d, J = 8.5), 6.93(2H, d, J = 8.7), 6.82 (2H, d, J = 8.5), 6.70 (1H, s), 4.13 (2H, t, J =
6.3Hz), 4.05 (2H, s), 3.79(2H, s), 2.93 (2H, t, J = 6.3Hz), 2.61~2.73 (4H, m), 1.04~1.13 (6H, m); ESI-MS (m/z):
465.0 (M+H+); IR (KBr): υ 3105, 2967, 2832, 1634, 1513, 1479, 1420, 1384, 1357, 1295, 1255, 1178, 1116, 1036,
878, 821, 758 cm-1; Anal. Calcd For C25H28N4O3S: C, 64.63; H, 6.07; N,12.06. Found: C, 64.54; H, 6.02; N, 12.11.
4.7.9. 6-(4-Methoxybenzyl)-3-{4-[2-(1-piperidinyl)ethoxy]phenyl}thiazolo[3,2-b]-1,2,4-triazin-7-one (7i)
1
This compound was obtained as a white crystal, 81.3% yield; mp: 171-173 °C; H-NMR (300 MHz, CDCl3): δ
7.47 (2H, d, J = 8.7Hz), 7.27 (2H, d, J = 8.4Hz), 6.94 (2H, d, J = 8.7Hz), 6.83 (2H, d, J = 8.4Hz), 6.72 (1H, s), 4.20
(2H, t, J = 5.9Hz), 4.05 (2H, s), 3.79 (3H, s), 2.86 (2H, t, J = 5.9Hz), 2.59 (4H, br), 1.64 (4H, br), 1.49 (2H, br); IR
(KBr): υ 3033, 2933, 1632, 1580, 1512, 1479, 1385, 1355, 1300, 1252, 1180, 1118, 1035, 820, 760 cm-1; Anal.
Calcd For C26H28N4O3S: C, 65.52; H, 5.92; N,11.76. Found: C, 65.44; H, 5.97; N, 11.71.
4.8 Biology Inhibition of AChE
The inhibitory potency against AChE was evaluated by means of Ellman’s test [13]. AChE stock solution was
prepared by dissolving human AChE 0.5 unit in 100 mM PBS buffer (pH 7.4). Tested target compounds (10 ꢁM)
were prepared in DMSO. The assay solution consisted of 100 mM PBS buffer (pH 7.4), with the addition of 10
mM 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB, Ellman’s reagent), AChE (5 ꢁL), drug (10 ꢁL), and 12.5 mM
acetylthiocholine iodide (ATCh) water solution. The final assay volume was 900 ꢁL. Incubate the reaction at 37ºC
for 15 min with continuous gentle shake. Add 50 mL ATCh and 50 mL DTNB. Incubate at 37ºC for about 20 min
with continuous gentle shake, Wait until the yellow color developed. Measure at 412 nm. Calculate the specific
inhibition rates.
4.9 Docking studies
The protein structure was prepared starting from the crystallographic complex of recombinant human AChE in
complex with donepezil (PDB ID: 4EY7) [14]. Donepezil and other non-protein molecules were deleted and only
the enzyme chain was retained. Hydrogen atoms and missing heavy atoms were added. Zero occupancy side chains,
polar hydrogen atoms, and the positions of asparagine and glutamine side chain amidic groups were optimized and
assigned the lowest energy conformation.
The Autodock software version 4 was used for the molecular docking process. The grids were constructed
around the proteins. The Lamarckian Genetic Algorithm method was used for the global optimum binding position
search. A number of 100 cycles of calculation were used in order to get a final binding position as accurate as
possible. All the compounds were docked into the active site of 4EY7. The complex of ligand-receptor was viewed
by PyMol program.
Acknowledgements
This work was supported by Scientific Research Fundation of Shijiazhuang University (No. XJPT007).
7

ACCEPTED MANUSCRIPT
References
[1] W. Xing, Y. Fu, Z. Shi, D. Lu, H. Zhang, Y. Hu, Eur. J. Med. Chem. 63 (2013) 95-103.
[2] A. Martorana, Z. Esposito, G. Koch, CNS Neurosci Ther. 16 (2010) 235-245.
[3] M. Catto, L. Pisani, F. Leonetti, O. Nicolotti, P. Pesce, A. Stefanachi, S. Cellamare, A. Carotti, Bioorg. Med.
Chem. 21 (2013) 146-152.
[4] E. A. Van der Zee, B. Platt, G. Riedel, Behav. Brain Res. 221 (2011) 583-587.
[5] F. Meng, F. Mao, W. Shan, F. Qin, L. Huang, X. Li, Bioorg. Med. Chem. 22 (2012) 4462-4466.
[6] G. Kryger, M. Harel, K.Giles, L.Toker, B. Velan, A. Lazar, C. Kronman, Acta Crystallogr. Sect. D. 56
(2000) 1385-1394
[7] P. Kapkováa, N. Stiefla, U. Süriga, Arch. Pharm. Pharm. Med. Chem. 336 (2003) 523-528.
[8] Z. Jin, L. Yang, S. J. Liu, J. Wang, C. Hu, Arch. Pharm. Res. 33 (2010) 1641-1649.
[9] I. Dorronsoro, D. Alonso, A. Castro, M. del Monte, E. García-Palomero, A. Martínez, Arch. Pharm. Med.
Chem. 338 (2005), 18-23.
[10] S. Liu, L. Yang, X. Liu, D. Wan, H. Lin, C. Hu, Lett. Drug. Des. Discov. 7 (2010), 5-8.
[11] S. Liu, L. Yang, Z. Jin, E. Huang, D Wan, H. Lin and C. Hu, ARKIVOC, X (2009) 333-348.
[12] H. Xu, Z. Jin, S. Liu, H. Liu, D. Wan, H. Lin, C. Hu, Chin. Chem. Lett. 23 (2012) 765-772.
[13] G. L. Ellman, K. D. Courtney, V. J. Andres, R. M. A. Featherstone, Biochem.Pharmacol. 7 (1961) 88.
[14] J. Cheung, M. J. Rudolph, F. Burshteyn, M. S. Cassidy, E. N. Gary, J. Love, M. C. Franklin, J. J. Height, J.
Med. Chem. 55 (2012) 10282-10286.
8

ACCEPTED MANUSCRIPT
Figure captions
Caption 1 Table 1. Inhibition of AChE Activities by the targets at 10ꢁM (n=2).
Caption 2 Fig. 1. Stucture of reported AChE inhibitors.
Caption 3 Fig. 2. Human AChE showing the relative disposition of catalytic (yellow), anionic binding (blue) and
peripheral (red) sites.
Caption 4 Fig. 3. Superimposed result between 1B41 (red) and 4EY7 (green).
Caption 5 Fig. 4. Superimposition of the best docking conformations of 7a (red), 7b (green), 7c (blue), 7d
(yellow), 7e (magenta), 7f (cyan), 7g (orange), 7h (salmon), and 7i (white) inside the gorge of 4EY7.
Caption 6 Fig. 5. Docking of compounds 7a-i to 4EY7.
Caption 7 Scheme 1. The synthetic route of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives.
9

ACCEPTED MANUSCRIPT
Table 1. Inhibition of AChE Activities by the targets at 10ꢁM (n=2)
No.
7a
R1
H
R2
R3
H
R4
H
Inhibition (%)
30.46
7b
OCH3
H
H
42.74
7c
7d
7e
H
OCH3
H
H
H
H
CH3
CH3
H
89.82
73.92
62.05
7f
H
H
H
H
H
68.74
79.36
7g
OCH3
7h
7i
OCH3
OCH3
H
H
H
H
50.95
54.67
100
huperzine-A
10

ACCEPTED MANUSCRIPT
Fig. 1. Structures of reported AChE inhibitors.
11

ACCEPTED MANUSCRIPT
Fig. 2. Human AChE showing the relative disposition of catalytic (yellow), anionic binding (blue) and
peripheral (red) sites.
12

ACCEPTED MANUSCRIPT
Fig. 3. Superimposed result between 1B41 (green) and 4EY7 (red).
13

ACCEPTED MANUSCRIPT
Fig. 4. Superimposition of the best docking conformations of 7a (red), 7b (green), 7c (blue), 7d (yellow), 7e
(magenta), 7f (cyan), 7g (orange), 7h (salmon), and 7i (white) inside the gorge of 4EY7. Only a part of residues of
4EY7 were showed as surface to reveal the binding gorge.
14

ACCEPTED MANUSCRIPT
15

ACCEPTED MANUSCRIPT
Fig. 5. Docking of compounds 7a-i to 4EY7.
16

ACCEPTED MANUSCRIPT
O
O
O
O
Ac2O, AcONa
acetone, H2O
refluxed, 10 h
HCl
OH
OH
R1
CHO
O
N
HN
CH3
CH2COOH
NHCOCH3
120 oC, 7 h
R1
refluxed, 10 h
O
R1
R1
CH3
2
1
3
4
1a R1 = H;
2a R1 = H; 2b R1 = OCH3
3a R1 = H; 3b R1 = OCH3
4a R1 = H; 4b R1 = OCH3
1b R1 = OCH3
OH
O
O
Cl
CH3
Cl
or
OH
+
N
HO
NH2NHCSNH2
NH
OH
CH3
N
N
N
N
ethaol
refluxed, 2 h
AcOH, AcONa
refluxed, 8-24 h
R1
O
N
S
H
S
S
O
N
R1
N
O
R1
5
6
6
5a R1 = H; 5b R1 = OCH3
6a, 6e R1 = H; 6b, 6f R1 = OCH3
6c R1 = H; 6d R1 = OCH3
substituted alkyl chloride K2CO3
acetone
refluxed, 24 h
R4
R3
R2
N
N
S
R1
O
N
7
O
7a R1 = H R2
=
O
7f R1 = R2 = R3 = H R4
=
R3 = R4 = H
N
N
O
O
O
O
7b R1= OCH3 R 2
7c R1 = R2 = H R3 = CH3 R4
7d R1 =OCH3 R2 = H R3 = CH3
=
O
R3 = R4 = H
7g R1 =OCH3 R2 = R3 = H R4
7h R1 =OCH3 R2 = R3 = H R4
7i R1 = OCH3 R2 =R3 = H R4
=
=
N
N
O
O
O
=
N
N
=
R4
=
O
N
N
O
O
7e R1 = R2 = R3 =H R4
=
N
Scheme 1. The synthetic route of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives
17

ACCEPTED MANUSCRIPT
·A series of novel 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were synthesized.
·Synthesized compounds were evaluated for their inhibitory activity by the classic Ellman test.
·Compounds 7c, 7d and 7g showed excellent inhibitory activity in the range from 73.92-89.82 %.
·Compound 7c, 7d and 7g can perfectly fit into the CAS and PAS simultaneously in the docking
model.