Synthesis and biological evaluation of 3,6-diaryl-7H-thiazolo[3,2-b] [1,2,4]triazin-7-one derivatives as acetylcholinesterase inhibitors

Article abstract of DOI:10.1007/s12272-010-1013-8

Acetylcholinesterase (AChE) inhibitors played an important role in developing a cure for Alzheimer' s disease. In order to study on the influence of modifications at different groups and side chains on the AChE inhibitory ability and the active sites of 7

Full text of DOI:10.1007/s12272-010-1013-8

Arch Pharm Res Vol 33, No 10, 1641-1649, 2010
DOI 10.1007/s12272-010-1013-8
Synthesis and Biological Evaluation of 3,6-diaryl-7H-thiazolo[3,2-b]
[1,2,4]triazin-7-one Derivatives as Acetylcholinesterase Inhibitors
Zhe Jin1,2, Liu Yang1,2, Si-Jie Liu1,2, Jian Wang1,2,Shuo Li3, Huang-Quan Lin3, David Chi Cheong Wan3, and
Chun Hu1,2
2
1Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, China, School of Pharmaceutical
3
Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China, and Department of Biochemistry, The Chi-
nese University of Hong Kong, Hong Kong SAR, China
(Received April 21, 2010/Revised June 18, 2010/Accepted June 30, 2010)
Acetylcholinesterase (AChE) inhibitors played an important role in developing a cure for
Alzheimer’ s disease. In order to study on the influence of modifications at different groups and
side chains on the AChE inhibitory ability and the active sites of 7
H-thiazolo[3,2-b][1,2,4]tri-
azin-7-one derivatives, fourteen 3,6-diaryl-7 -thiazolo[3,2- ][1,2,4]triazin-7-one derivatives
H
b
were designed and synthesized. The study of AChE inhibitory activity was carried out using
the Ellman colorimetric assay with huperzine-A as the positive control drug. Most of the tar-
get compounds exhibited more than 50% inhibition at 10 µM. Some target compounds showed
strong inhibition against AChE. The molecular fields analysis and preliminary structure-
activity relationships are discussed.
Key words: Acetylcholinesterase, Inhibitor, Heterocycle, Thiazole, 1,2,4-Triazine, Molecular
docking, Molecular fields analysis
In recent years, a series of crystal structures of the
Torpedo californica AChE (TcAChE) and human AChE
INTRODUCTION
Alzheimer’s disease (AD) is the most common age-
(hAChE) have been reported. In TcAChE, the struc-
related neurodegenerative disorder (LaFerla et al., ture indicated that there is a deep, narrow gorge ap-
2007). In the United States, an estimated 4.5 million proximately 200 nm long on the surface of the enzyme,
individuals are currently suffering from AD (Hebert called the catalytic active site (CAS), which was as-
et al., 2003). According to a survey released by the signed to the Ser-His-Glu catalytic triad (Ser200-
Shanghai Mental Health Centre in the 1990s, approx- His440-Glu327). In the middle of the gorge, there are
imately 4.69% people in Shanghai China suffered from 5 amino acid residues, including Trp84, Tyr130, Glu199,
AD (Wang et al., 2002). As a result, AD treatment and Phe330, and Tyr442, which comprise the anionic bind-
care constitutes a major social and health problem ing site. At the entrance of the aromatic gorge, there
(Ceballos and Guzman, 2005).
is a peripheral anionic site (PAS), including Tyr70,
To date, acetylcholinesterase (AChE) inhibitors are Asp72, Tyr121, Glu278, Trp279, and Tyr334 residues
the main type of drugs used to treat AD. Current (Sussman et al., 1991; Khalid et al., 2004).
medications that have received FDA approval for the
Previously, the interaction between 4 inhibitors,
treatment of AD include AChE inhibitors, such as tacrine (Davis and Powchik, 1995; Shao and Li, 2004),
tacrine, donepezil, rivastigmine, galanthamine, and donepezil (Kryger et al., 1999; Tsuno, 2009), rivastig-
an NMDA receptor antagonist, memantine.
mine (Polinsky, 1998; Grossberg, 2005), huperzine A
(Raves et al., 1997; Bai et al., 2000), and AChE, have
been investigated by flexible docking and X-ray diffrac-
tion crystal structure of the AChE inhibitor-AChE
complex. These results showed that some AChE inhi-
bitors, such as donepezil, interact with the PAS and
Correspondence to: Chun Hu, Department of Organic Chemis-
try, Shenyang Pharmaceutical University, Shenyang 110016,
China
Fax: 86-24-23986050
E-mail: chunhu@syphu.edu.cn
1641

1642
Z. Jin et al.
the anionic binding site of AChE, while other inhi- spray voltage, acetonitrile was used as the solvent.
bitors, such as rivastigmine, interact with the CAS of 1H-NMR spectra were obtained in DMSO-d6 or CDCl3
AChE. Recent studies have suggested that dual-bind- on a Bruker spectrometer operating at 300 MHz or
ing inhibitors not only increase the inhibitory activity 600 MHz. The IR spectra were determined using a
and selectivity of AChE but also prevent the aggre- Bruker AFS55 spectrometer.
gation of toxic β-amyloid (Aβ) (Inestrosa et al., 1996;
Reyes et al., 1997). Hence, dual-binding AChE inhibi-
tors, which interact with the PAS and the CAS of
AChE, are hot topics in research on AChE inhibitors.
Synthesis of 2-aryl-2-oxoacetic acids (2)
A mixture of styrene derivatives ( ) (0.06 mol), NaOH
(0.06 mol), and KMnO4 (0.09 mol) were dissolved in
1
Our previous work has shown 5H-thiazolo[3,2-a]pyr- H2O (500 mL) in a 1000 mL round bottom flask, heated
imidine derivatives exhibited AChE inhibitory acti- to 50oC, and stirred for 20 min. Ethanol (20 mL) was
vity, and the molecular docking exhibited the com- then dropped slowly into the flask and stirred for 15
pounds interacted with the PAS of AChE (Zhi et al., min. The mixture was filtered, washed with hot water,
2008). 6-Arylmethyl-7H-thiazolo[3,2-b][1,2,4]triazin-7- concentrated, acidified with concentrated HCl, and
one derivatives also exhibited AChE inhibitory acti- extracted with ethyl acetate (25 mL
×
4). The organic
vity, and the molecular docking exhibited the com- layer was dried and distilled to give 2-aryl-2-oxoacetic
pounds interacted with the PAS and the CAS of AChE acid.
(Liu et al., 2009).
In order to study the the structure-AChE inhibitory 2-(4-Chlorophenyl)-2-oxoacetic acid (2a)
activity relationships and the active sites of 7
thiazolo[3,2- ][1,2,4]triazin-7-one derivatives, a new chromatography (TLC), spreading solvents were acet-
series of 3,6-diaryl-7 -thiazolo[3,2- ][1,2,4]triazin-7- ate/n-hexane (V/V = 2/1), Rf = 0.32.
H-
White solid, 60% yield. Only one spot on thin layer
b
H
b
one derivatives were designed and synthesized, and
their inhibitory activity was determined.
2-(4-Bromophenyl)-2-oxoacetic acid (2b)
White solid, 76% yield. Only one spot on TLC, spread-
ing solvents were acetate/n-hexane (V/V = 2/1), Rf =
0.30.
MATERIALS AND METHODS
Molecular docking
The crystal structure of the
2) complex was selected from the Protein Data Bank. azin-5(2
The inhibitor structure and water molecules were A mixture of
h
AChE (1B41/fasciculin- Synthesis of 6-aryl-3-thioxo-3,4-dihydro-1,2,4-tri-
)-ones (3)
0.20 mol, thiosemicarbazide (0.25 mol),
H
2
deleted from the complex, the missing residues were ethanol (20 mL), and concentrated NaOH in aqueous
built in, and the polar hydrogen atoms were added to solution (15 mL) was refluxed for 7 h, cooled to room
the amino acid residues. Molecular docking was carried temperature, and adjusted to pH 2 with HCl. The
out using the Molegro Virtual Docker (MVD) (Thomsen solid was collected to give 6-(4-halophenyl)-3-thioxo-
and Christensen, 2006). The docking algorithm was 3,4-dihydro-1,2,4-triazin-5(2
H)-one.
set at 1,500 maximum iterations with a simplex evo-
lution population size of 50 and a minimum of 10 6-(4-Chlorophenyl)-3-thioxo-3,4-dihydro-1,2,4-tri-
runs. Schematic diagrams of the interactions between azin-5(2
H)-one (3a)
1B41 and the small molecules were analyzed using White powder, 72% yield; mp: 279-282oC.
MVD.
6-(4-Bromophenyl)-3-thioxo-3,4-dihydro-1,2,4-tri-
azin-5(2H)-one (3b)
Chemistry
All reagents and solvents were purchased from Yellowish powder, 67% yield; mp: 272-275oC.
common commercial suppliers and were used without
further purification. Melting points were determined Synthesis of 3, 6-diaryl-7H-thiazolo[3,2-b][1,2,4]
on a Koffler hot-plate apparatus and were uncorrect- triazin-7-ones (4)
ed. The C, H, and N element analyses were performed A mixture of 6-aryl-3-thioxo-3,4-dihydro-1,2,4-triazin-
on a Thermo Flash EA1112 elemental analyzer. Mass 5(2 )-one (10 mmol), substituted phenacyl chloride
H
spectra (MS) were obtained using an electronic impact (10 mmol), and AcOH (20 mL) was heated under reflux
(EI) ion source at 70 eV in an Agilent spectrometer for 12 h. The solution was then cooled to room temper-
(with direct insertion probe) or with an electrospray ature and filtered. The solid was collected and recry-
(ESI) ion source in a Waters spectrometer at 3.5 kV stallized from absolute ethanol.

7H-Thiazolo[3,2-b][1,2,4]triazin-7-one Derivatives as Acetylcholinesterase Inhibitors
1643
3-(4-Methylphenyl)-6-phenyl-7
[1,2,4]triazin-7-one (4a)
H
-thiazolo[3,2-
b
]
DMSO-d6):
δ 9.88 (1H, s), 7.98 (2H, d, J = 9.0 Hz), 7.50
(1H, d, J = 7.8 Hz), 7.48 (2H, d, J = 7.8 Hz), 7.46 (1H,
Yellowish powder, 66% yield; mp: 221-223oC; ESI-MS s), 6.91 (1H, d, J = 7.8 Hz), 2.50 (3H, s); IR (KBr, cm-1):
m/z
(relative intensity): 320.0 ([M+H]+, 17), 341.9 ([M
+Na]+, 62), 661.0 ([2M+Na]+, 100); 1H-NMR (300 MHz, calcd. for C18H13O2N3S (%): C, 64.46; H, 3.91; N, 12.53;
CDCl3): 8.23 (2H, dd, J = 6.5 Hz), 7.61 (2H, d, J = Found (%): C, 64.38; H, 3.86; N, 12.48.
υ 3048, 1774, 1635, 1545, 1466, 1282, 689, 527; Anal.
δ
8.13 Hz), 7.48 (2H, m), 7.46 (2H, d, J = 7.7 Hz), 7.34
(2H, d, J = 7.8 Hz), 6.84 (1H, s), 2.46 (3H, s); IR (KBr, 6-(4-Chlorophenyl)-3-phenyl-7H-thiazolo[3,2-b][1,
cm-1):
υ
3069, 1638, 1538, 1468, 1384, 1350, 542; Anal. 2,4]triazin-7-one (4f)
calcd. for C18H13ON3S (%): C, 67.69; H, 4.10; N, 13.16; White powder, 39% yield; mp: 283-285oC; ESI-MS
S, 10.04; Found (%): C, 67.63; H, 4.05; N, 13.13.
(relative intensity): 339.9 ([M+H]+, 100), 341.9 ([M+2+
H]+, 34), 361.9 ([M+Na]+, 48), 363.9 ([M+2+Na]+, 18);
1H-NMR (300 MHz, DMSO-d6):
8.02 (2H, d, J = 8.4
Hz), 7.76 (5H, m), 7.71 (2H, d, J = 8.4 Hz), 7.66 (1H, s);
3063, 1632, 1541, 1469, 1384, 1353,
(relative intensity): 339.9 ([M+H]+, 13), 361.9 ([M+ 840, 759; Anal. calcd. for C17H10ON3SCl (%): C, 60.09;
Na]+, 100), 700.8 ([2M+Na]+, 16); 1H-NMR (300 MHz, H, 2.97; N, 12.37; Found (%): C, 60.03; H, 2.94; N,
CDCl3): 8.20 (2H, d, J = 7.8 Hz), 7.66 (2H, d, J = 8.4 12.33.
Hz), 7.53 (2H, d, J = 8.4 Hz), 7.47 (2H, d, J = 7.2 Hz),
6.90 (1H, s); IR (KBr, cm-1):
3067, 1642, 1537, 1471, 3-(4-Chlorophenyl)-6-(4-bromophenyl)-7
1383, 1353, 547; Anal. calcd. for C17H10ON3SCl (%): C, [3,2- ][1,2,4]triazin-7-one (4g)
60.09; H, 2.97; N, 12.37; Found (%): C, 60.03; H, 2.94; Yellow needle crystal, 12% yield; mp: 314-316oC; ESI-
m/z
3-(4-Chlorophenyl)-6-phenyl-7
[1,2,4]triazin-7-one (4b)
H
-thiazolo[3,2-
b
]
δ
Yellowish powder, 59% yield; mp: 269-271oC; ESI-MS IR (KBr, cm-1):
υ
m/z
δ
υ
H-thiazolo
b
N, 12.33.
MS m/z
(relative intensity): 417.7 ([M+H]+, 29), 419.8
([M+2+H]+, 40), 421.8 ([M+4+H]+, 13); 439.7 ([M+Na]+,
63), 441.7 ([M+2+Na]+, 100); 455.7 ([M+K]+, 21), 457.7
6-Phenyl-3-(4-tert-butylphenyl)-7
H-thiazolo[3,2-b]
1
[1,2,4]triazin-7-one (4c)
([M+2+K]+, 31), 459.7 ([M+4+K]+, 16); H-NMR (300
Yellowish powder, 62% yield; mp: 216-217oC; ESI-MS MHz, DMSO-d6):
δ
8.02 (2H, d), 7.92 (2H, d), 7.74 (4H,
3443, 3079, 1642,
1
m/z
(relative intensity): 400.0 ([M+K]+, 69); H-NMR m), 7.53 (1H, s); IR (KBr, cm-1):
υ
(600 MHz, CDCl3):
δ 8.21 (2H, m), 7.66 (2H, d, J = 7.8 1535, 1466, 1384, 1279, 835, 520; Anal. calcd. for
Hz), 7.55 (2H, d, J = 8.4 Hz), 7.49 (1H, d, J = 7.8 Hz), C17H9ON3SClBr (%): C, 48.77; H, 2.17; N, 10.04; Found
7.45 (2H, m), 6.84 (1H, s), 1.40 (9H, s); IR (KBr, cm-1): (%): C, 48.70; H, 2.14; N, 9.99.
υ
3087, 2961, 1637, 1543, 1469, 1384, 546; Anal. calcd.
for C21H19ON3S (%): C, 69.78; H, 5.30; N, 11.63; Found 6-(4-Bromophenyl)-3-(4-methylphenyl)-7
H-thiazolo
(%): C, 69.71; H, 5.27; N, 11.58.
[3,2-b][1,2,4]triazin-7-one (4h)
Yellow powder, 31% yield; mp: 283-286oC; ESI-MS
m/
3-(4-Methoxy-3-methylphenyl)-6-phenyl-7
H
-thia-
z
(relative intensity): 398.0 ([M+H]+, 62), 400.0 ([M+2
+H]+, 58), 420.0 ([M+Na]+, 79), 422.0 ([M+2+Na]+, 100),
zolo[3,2-
Yellowish powder, 61% yield; mp: 234-236oC; ESI-MS 436.0 ([M+K]+, 60), 438.0 ([M+2+K]+, 82); 1H-NMR (300
(relative intensity): 349.9 ([M+H]+, 14), 372.0 ([M+ MHz, DMSO-d6):
8.02 (2H, d, J = 8.4 Hz), 7.87 (2H,
Na]+, 100), 721.0 ([2M+Na]+, 22); 1H-NMR (600 MHz, d, J = 8.4 Hz), 7.72 (2H, d, J = 8.4 Hz), 7.53 (1H, s), 7.36
CDCl3): 8.22 (2H, m), 7.54 (1H, dd, J1 = 8.4 Hz, J2 3425,
(2H, d, J = 8.4 Hz), 2.39 (3H, s); IR (KBr, cm-1):
b][1,2,4]triazin-7-one (4d)
m/z
δ
δ
=
υ
2.4 Hz), 7.48 (2H, d, J = 7.2 Hz), 7.44 (2H, dd, J1 = 7.8 3066, 1635, 1539, 1466, 837, 815, 550, 524; Anal. calcd.
Hz, J2 = 1.2 Hz), 6.95 (1H, d, J1 = J2 = 8.4 Hz), 6.76 for C18H12ON3SBr (%): C, 54.28; H, 3.04; N, 10.55;
(1H, s), 3.91 (3H, s), 2.29 (3H, s); IR (KBr, cm-1):
υ
Found (%): C, 54.21; H, 3.00; N, 10.50.
3135, 2919, 1684, 1567, 1469, 1385, 1329, 562; Anal.
calcd. for C19H15O2N3S (%): C, 65.31; H, 4.33; N, 12.03; 6-(4-Chlorophenyl)-3-(4-methoxyphenyl)-7
H-thia-
Found (%): C, 65.26; H, 4.28; N, 11.97.
zolo[3,2-b][1,2,4]triazin-7-one (4i)
Yellow powder, 36% yield; mp: 291-293oC; ESI-MS
m/
3-(4-Hydroxy-3-methylphenyl)-6-phenyl-7
H-thia-
z
(relative intensity): 370.1 ([M+H]+, 67), 392.1 ([M+
Na]+, 100), 394.0 ([M+2+Na]+, 39), 408.0 ([M+K]+, 55),
zolo[3,2-
Yellowish powder, 60% yield; mp: 201-203oC; ESI-MS 410.0 ([M+2+K]+, 27); 1H-NMR (300 MHz, DMSO-d6):
(relative intensity): 336.1 ([M+H]+, 57), 358.2 ([M+
8.10 (2H, d, J = 8.7 Hz), 7.73 (2H, d, J = 8.7 Hz),
Na]+, 100), 693.4 ([2M+Na]+, 25); 1H-NMR (600 MHz, 7.57 (2H, d, J = 8.7 Hz), 7.48 (1H, s), 7.11 (2H, d, J =
b][1,2,4]triazin-7-one (4e)
m/z
δ

1644
Z. Jin et al.
8.7 Hz), 3.36 (3H, s); IR (KBr, cm-1):
υ 3443, 3067, H, 4.41; N, 13.73.
1645, 1538, 1466, 1384, 1254, 837, 524; Anal. calcd. for
C18H12O2N3SCl (%): C, 58.46; H, 3.27; N, 11.36; Found 3-{4-[2-Oxo-2-(4-morpholinyl)ethoxy]phenyl}-6-
(%): C, 58.41; H, 3.24; N, 11.31.
phenyl-7
Yellowish powder, 60% yield; mp: 129-130oC; ESI-MS
(relative intensity): 449.3 ([M+H]+, 22), 471.2 ([M+
Na]+, 100); 1H-NMR (600 MHz, DMSO-d6):
8.03 (2H,
Yellowish powder, 60% yield; mp: 171-172oC; ESI-MS d, J = 7.2 Hz), 7.71 (2H, d, J = 9.0 Hz), 7.51 (1H, d,
(relative intensity): 343.9 ([M+Na]+, 100), 664.8
7.8 Hz), 7.48 (2H, d, J = 7.8 Hz), 7.47 (1H, s), 7.09
([2M+Na]+, 14); 1H-NMR (300 MHz, DMSO-d6):
9.95 (2H, d, J = 8.4 Hz), 4.94 (2H, s), 3.62 (2H, t), 3.57 (2H,
(2H, d, J = 8.4 Hz), 8.06 (2H, dd, J1 = 7.2 Hz, J2 = 1.5 t), 3.45 (4H, d, J = 13.2 Hz); IR (KBr, cm-1):
3114,
Hz), 7.61 (2H, d, J = 8.7 Hz), 7.49 (3H, m), 7.39 (1H, 2971, 1635, 1545, 1469, 1384, 1351, 551; Anal. calcd.
s), 6.92 (1H, s), 6.89 (1H, s); IR (KBr, cm-1):
3118, for C23H20O4N4S (%): C, 61.60; H, 4.49; N, 12.49; Found
H-thiazolo[3,2-b][1,2,4]triazin-7-one (5b)
3-(4-Hydroxyphenyl)-6-phenyl-7
H
-thiazolo[3,2-
m/z
b][1,2,4]triazin-7-one (4j)
δ
J
m/z
=
δ
υ
υ
1720, 1610, 1507, 1471, 1384, 1325, 548; Anal. calcd. (%): C, 61.52; H, 4.45; N, 12.45.
for C17H11O2N3S (%): C, 63.54; H, 3.45; N, 13.08;
Found (%): C, 63.49; H, 3.40; N, 13.04.
3-{4-[2-(4-Piperidyl)ethoxy]phenyl}-6-phenyl-7
thiazolo[3,2- ][1,2,4]triazin-7-one (5c)
Yellowish powder, 65% yield; mp: 151-153oC; ESI-MS
(relative intensity): 433.3 ([M+H]+, 100); 1H-NMR
(600 MHz, DMSO-d6): 8.05 (2H, d, J = 6.6 Hz), 7.71
(relative intensity): 414.0 ([M+H]+, 85), 416.0 ([M+2+ (2H, d, J = 9.0 Hz), 7.51 (1H, d, J = 7.2 Hz), 7.48 (2H,
H]+, 77), 436.0 ([M+Na]+, 100), 438.0 ([M+2+Na]+, 81), d, J = 7.8 Hz), 7.46 (1H, s), 7.12 (2H, d, J = 9.0 Hz),
452.0 ([M+K]+, 40), 454.0 ([M+2+K]+, 47); 1H-NMR (300 4.14 (2H, t, J1
J2 = 6.0 Hz), 2.68 (2H, m), 2.48 (4H,
MHz, DMSO-d6): 3070,
8.02 (2H, d, J = 8.7 Hz), 7.72 (4H, m), 1.50 (4H, m), 1.38 (2H, s); IR (KBr, cm-1):
m), 7.48 (1H, s), 7.11 (2H, d, J = 8.7 Hz), 3.84 (3H, s); 2931, 1636, 1544, 1473, 1384, 1351, 549; Anal. calcd.
IR (KBr, cm-1):
3423, 3068, 1643, 1586, 1538, 1465, for C24H24O2N4S (%): C, 66.64; H, 5.59; N, 12.95;
H-
b
6-(4-Bromophenyl)-3-(4-methoxyphenyl)-7H-thi-
azolo[3,2-
b
][1,2,4]triazin-7-one (4k)
m/z
Yellow powder, 26% yield; mp: 289-291oC; ESI-MS
m/
δ
z
=
δ
υ
υ
1384, 1278, 1255, 1179, 1078, 1010, 835, 553, 521; Found (%): C, 66.57; H, 5.57; N, 12.91.
Anal. calcd. for C18H12O2N3SBr (%): C, 52.19; H, 2.92;
N, 10.14; Found (%): C, 52.10; H, 2.88; N, 10.09.
Inhibition of AChE
The compounds dissolved in DMSO were tested
][1,2,4] for AChE inhibitory activity by the Ellman assay
(Elsinghorst et al., 2006). The Ellman assay was
Synthesis of 3,6-diaryl-7
triazin-7-ones (5)
H-thiazolo[3,2-b
A mixture of 3-(4-hydroxyphenyl)-6-aryl-7
[3,2-b][1,2,4]triazin-7-one (10 mmol), 2-chloro-
ethylacetamide or 2-chloro-1-(piperidin-1-yl)ethanone 7.4) was mixed with 0.1 unit of
or -benzyl-2-chroloacetamide (10 mmol), potassium 1682). After a 15 min pre-incubation, equal amounts
H
-thiazolo performed in 96 well plates as follows: the compound
- di- in assay buffer (100 mM sodium phosphate buffer, pH
AChE (Sigma, C-
N,N
h
N
carbonate (50 mmol), potassium iodide (1 mmol), and of 12.5 mM of acetylthiocholine iodide (ATCI) (Sigma,
acetone (25 mL) was heated under reflux for 24 h. A-5751) and 10 mM 5’5-dithio-bis-(2-nitrobenzoate)
Then the mixture was poured into H2O (100 mL) and DTNB (Sigma, D-8130) were added, and then 100
stirred for 12 h. The solid was collected and recry- of the mixture was added to individual wells. After a
µL
stallized from absolute ethanol.
20 min incubation with the substrate, the optical
densities were measured in a 96-well plate reader at
415 nm. The optical density was inversely propor-
tional to the inhibitory activity. Percentage inhibitions
3-[4-(2-Dimethylamino-2-oxoethoxy)phenyl]-6-
phenyl-7H-thiazolo[3,2-b][1,2,4]triazin-7-one (5a)
Yellowish powder, 70% yield; mp: 231-233oC; ESI-MS were calculated as compared to control. Data analysis
m
/
z
(relative intensity): 407.1 ([M+H]+, 16), 429.2 ([M+ was performed with Sigma Plot 2001 software. In-
Na]+, 100), 835.3 ([2M+Na]+, 15); 1H-NMR (600 MHz, hibitory effects were expressed as the percentage of
DMSO-d6): 8.04 (2H, d, J = 7.8 Hz), 7.71 (2H, m), inhibition.
δ
7.53 (1H, d, J = 7.2 Hz), 7.49 (2H, d, J = 7.8 Hz), 7.47
(1H, s), 7.08 (2H, m), 4.91 (2H, s), 3.00 (3H, s), 2.85
RESULTS AND DISCUSSION
(3H, s); IR (KBr, cm-1):
υ 3135, 2926, 1667, 1592, 1471,
1386, 1355, 1317, 557; Anal. calcd. for C21H18O3N4S
(%): C, 62.06; H, 4.46; N, 13.78; Found (%): C, 61.99;
Docking studies
Comparison of the three-dimensional structures of

7H-Thiazolo[3,2-b][1,2,4]triazin-7-one Derivatives as Acetylcholinesterase Inhibitors
1645
TcAChE (1EVE) and
degree of similarity, especially with regard to the active sites of
active sites. The most significant difference between coded as follows: the amino acid residues of CAS are
the active sites of TcAChE and AChE is that the shown in gray, the residues of anionic binding site are
Phe330 of TcAChE was replaced with a Tyr337 of shown in green, and the residues of PAS are shown in
AChE. Another difference is that the sequence num- yellow. There was a high degree of overlap between all
ber of some protein residues; for example, Ser200 of of the target compounds, especially the scaffold moiety,
TcAChE was replaced by Ser203 of AChE (Sussman the active sites of the target compounds were similar.
et al., 1991; Kryger et al., 1999) (Table I). The long side chains of the compounds 5a 5c were
AChE and extraordinary
hAChE (1B41) revealed a high
All of the target compounds were overlapped in the
hAChE (Fig. 1). The residues were color
h
h
h
-
The action sites of the various inhibitors and AChE also able to interact with
are also different (Table II). Tacrine was shown to in- active sites.
h
teracts with the CAS and the PAS of AChE (Shao and
Li, 2004), donepezil with the PAS and the anionic between 3-(4-hydroxy-3-methylphenyl)-6-phenyl-7
binding site of AChE (Kryger et al., 1999), rivastig- thiazolo[3,2- ][1,2,4]triazin-7-one (4e) and AChE,
According to the predicted docking interactions
H-
b
h
mine with the CAS of AChE (Polinsky, 1998), and hydrogen bonds (represented as green dotted lines,
huperzine A with the CAS and the anionic binding Fig. 2) occur between the carbonyl group at the C7
site of AChE (Raves et al., 1997).
position, the C8 position of the parent nucleus of com-
Table I. Differences in the protein residues of TcAChE and
h
AChE
Active Site
Active Site
TcAChE
hAChE
TcAChE
hAChE
Ser200
Glu327
His440
Ser203
Glu334
His447
Tyr70
Asp72
Tyr121
Tyr72
Asp74
Tyr124
Catalysis active
site (CAS)
Glu278
Trp279
Tyr334
Glu285
Trp286
Tyr341
Trp84
Trp86
Peripheral anionic
site (PAS)
Tyrl30
Glu199
Phe330
Tyr442
Tyr133
Glu202
Tyr337
Tyr449
Anionic binding
site
Table II. Different inhibitors interacted with different active sides of the AChE
AChE
Inhibitor
Protein Residues
Active Sites
CAS
AChE Inhibitor
Rivastigmine
Protein Residues
Active Sites
His440
Ser200
CAS
Tacrine
Asp72
PAS
His440
CAS
Trp279
PAS
Huperzine A
Trp84, Tyr130
Glu199, Phe330
Donepezil
Anionic binding site
Trp84, Phe330
Anionic binding site
Fig. 1. Docking model of the target compounds superimpos- Fig. 2. Docking model of compound 4e in the active sites of
ed in the active sites of AChE AChE
h
h

1646
Z. Jin et al.
Chemistry
The target compounds 4a
-k
were obtained in satis-
factory yields, and the synthetic pathways are describ-
ed in Scheme 1.
The target compounds 3-substituted aryl 7 -thia-
H
zolo[3,2- ]-1,2,4-triazin-7-one derivatives 5a-c were
b
obtained with the classical Williamson reaction shown
in Scheme 2.
The general procedure, described below, was de-
veloped for the generation of synthetic target com-
pounds. The styrene derivatives (
with yields from 60 to 73% in accordance with the
literature (Marvel and Schertz, 1943). Compound
reacted with the KMnO4 alkaline aqueous solution to
produce keto acids . Compound was prepared by a
reaction of compound with thiosemicarbazide in a
1) were synthesized
1
Fig. 3. Docking model of compound 5a in the active sites of
2
3
hAChE
2
sodium hydroxide aqueous solution. The target com-
pound 4e and the Tyr124 of
hAChE, between the N5 pounds 4a-k were obtained by a reaction of compound
position of the parent nucleus and the Tyr337 residue,
3 with substituted phenacyl chlorides in acetic acid.
and between the hydroxyl group at the aromatic ring The target compounds 5a-c were prepared with the
at the C3 position of the parent nucleus and Tyr 449. Williamson reaction shown in Scheme 2 (Ahmad et
The protein residue Tyr124 was shown to be located al., 1987; Liu et al., 1992; Ramadan et al., 2005).
in the peripheral anionic site (PAS) and Tyr337 and
The chemical structures of all target compounds
Tyr449 in the anionic binding site (Table I). Com- synthesized were fully characterized by mass spectra
pound 4e is thought to interact with the PAS and the (MS), element analysis, 1HNMR, and infrared spectra
anionic binding site of
hAChE.
data.
Similarly, there are predicted hydrogen bonds
between the parent nucleus of 3-[4-(2-dimethylamino-
Inhibition of AChE
2-oxoethoxy)phenyl]-6-phenyl-7
triazin-7-one (5a) and Tyr124, Tyr337 of
3) and the parent side chains of compound 5a and was tested three times (n = 3), while shaking for 20
Ser203 of AChE. It was also determined that the min. Inhibition of AChE activity by the positive
protein residue Ser203 is located in the catalysis control, huperzine-A, (10 M; n = 3) was 100%.
active site (CAS), Tyr124 is located in the PAS, and The compounds 4e and 4j, which have a hydroxyl
Tyr337 is located in the anionic binding site. Hence group, exhibited a more potent inhibitory effect than
the target compound 5a can interact with the CAS, 4a because the hydroxyl group was able to form a
the PAS, and the anionic binding site of AChE. hydrogen bond with Tyr449 of the AChE receptor
H-thiazolo[3,2-b][1,2,4]
The biological activity of
hAChE (0.5 units) was
hAChE (Fig. tested against the target compounds. Each compound
h
h
µ
-d
h
Scheme 1. The synthetic route of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives

7H-Thiazolo[3,2-b][1,2,4]triazin-7-one Derivatives as Acetylcholinesterase Inhibitors
1647
Scheme 2. The synthetic route of 3-substituted aryl 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives
Table III. Inhibition of AChE activity by the target compounds
No.
Inhibition (%)
No.
Inhibition (%)
No.
Inhibition (%)
4a
4b
4c
4d
4e
38.49
51.90
51.06
49.43
65.51
4f
4g
4h
4i
68.18
75.30
70.45
82.48
61.27
4k
5a
5b
5c
74.75
68.45
63.92
60.13
4j
The biological activity of hAChE (0.5 units) was tested against the target compounds. Each compound was tested three
times (n=3), while shaking for 20 min. Inhibition of
was 100%.
hAChE activity by the positive control, huperzine-A, (10 µM; n=3)
(Fig. 2). The compounds 5a
chains at the phenyl ring at the C3 position of the shown by the field point patterns (Fig. 5), some target
parent nucleus, were more potent than 4a because compounds, of which the inhibitory activities against
the long side chains of 5a were able to form a
-c, which have long side the large points indicate a strong interaction. As
-d
-c
hydrogen bond with Ser203 located in the CAS of the
AChE receptor (Fig. 3). 4f-i and 4k also had a strong
inhibitory effect, possibly due to the halogen atoms.
Molecular force field analysis
The Field Templater software and the Field Align
software were used for analyzing the molecular force
fields of the target compounds. The field point tem-
plate was built from compounds 4g
,
4h, 4i, 4k, and 5a
using the Field Templater (Fig. 4)
.
These five target
compounds showed a stronger inhibitory activity
against AChE than the other compounds. Different
color balls indicated the field points, which were color
coded as follows: negative ionic fields are shown in
blue, positive ionic fields are shown in red, van der
Waal’s surface is shown in yellow, and hydrophobic
fields are shown in orange. The size of the points
indicates the potential strength of the interactions;
Fig. 4. Field point template of compounds 4g
and 5a
, 4h, 4i, 4k,

1648
Z. Jin et al.
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Fig. 5. Field point patterns of some target compounds
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These phenomena showed that the CAS, the PAS
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long side chains at the phenyl ring at the C3 position,
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ACKNOWLEDGEMENTS
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This work was supported by the National Science
Foundation of China (NFSC) for the grant No.
21072130. The authors would like to thank Molegro
ApS and Cresset BioMolecular Discovery Ltd. for kindly
providing us with a free evaluation of their software
packages. The authors would also like to thank Ms.
He Zhu from Shandong University for acquiring the
NMR spectra data.
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