Synthesis, β-catenin translocation capability and ALP activation activity of 7h-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives

Article abstract of DOI:10.2174/1573406413666171002121443

Background: Osteoporosis (OP) is a common bone disease, most often diagnosed in post-menopausal women. The majority of OP treatments are focused on manipulation of the patient's hormone levels, therefore, they are associated with significant adverse effects. Objective: The study aimed to design, synthesize and evaluate the β-Catenin translocation capability and the alkaline phosphatase (ALP) activation activity of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives. Method: The styrene derivatives were synthesized as raw materials, followed by oxidation and condensation reactions, in which 6-aryl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one derivatives (1) were obtained. The 3,6-diaryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-ones (2) were obtained by a condensation reaction of compound 1 with substituted phenacyl chlorides in acetic acid. The target compounds 3,6-diaryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-ones (3a-3c) were prepared by compound 2 with substituted alkyl chloride by Williamson reaction. As to 6-benzyl-3-aryl-7H-thiazolo[3,2- b]-1,2,4-triazin-7-one derivatives as the target compounds, the benzaldehyde and acetylglycine used as raw materials, followed by Erlenmeyer-Pl?chl reaction, condensation reaction, hydrolysis reaction, condensation reaction, 6-benzyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one derivatives were obtained, and were converted to the target compounds 6-benzyl-3-(hydroxylaryl)-7Hthiazolo[ 3,2-b]-1,2,4-triazin-7-one derivatives (5a-5d) using reaction with substituted β-phenacyl chlorides. Finally, Williamson reaction were used to yield 6-benzyl-3-aryl-7H-thiazolo[3,2-b]- 1,2,4-triazin-7-ones as target compounds (6a-6e). The β-Catenin translocation capability and the ALP activation activity were tested, and the glycogen synthase kinase-3 (GSK-3) inhibition was simulated by molecular docking. Results: Fourteen 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were synthesized and characterized by mass spectra, proton NMR and infrared spectra, the β-Catenin translocation capability and the ALP activation activities of the target compounds were tested and calculated. The EC50 value of the ALP activation activity of 6-(4-chlorobenzyl)-3-{4-[(2-dimethylamino)-2- oxoethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (6b) was 11.283 μM. The molecular docking results have showed that the target compounds would be GSK-3 inhibitors. Conclusion: Based on the results of the biological activity test, the target compounds have exhibited the β-Catenin translocation capability and the ALP activation activity.

Full text of DOI:10.2174/1573406413666171002121443

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Medicinal Chemistry, 2018, 14, 67-73
RESEARCH ARTICLE
ISSN: 1573-4064
eISSN: 1875-6638
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Synthesis, ꢀ-catenin Translocation Capability and ALP Activation Activity
of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one Derivatives
Shicheng Hou1, Henan Xu1, Jianshu Hu2, Jian Hou1, Yan Wang2, Zhe Jin1,*, David C.C. Wan2 and
Chun Hu1,*
1Key Laboratory of Structure-based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical Uni-
versity, Shenyang 110016, P.R. China; 2School of Biomedical Sciences, Faculty of Medicine, The Chinese University of
Hong Kong, Hong Kong SAR, P.R. China
Abstract: Background: Osteoporosis (OP) is a common bone disease, most often diagnosed in
post-menopausal women. The majority of OP treatments are focused on manipulation of the pa-
tient’s hormone levels, therefore, they are associated with significant adverse effects.
Objective: The study aimed to design, synthesize and evaluate the ꢀ-catenin translocation capabil-
ity and the alkaline phosphatase (ALP) activation activity of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one
derivatives.
Method: The styrene derivatives were synthesized as raw materials, followed by oxidation and
condensation reactions, in which 6-aryl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one derivatives
(1) were obtained. The 3,6-diaryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-ones (2) were obtained by a
condensation reaction of compound 1 with substituted phenacyl chlorides in acetic acid. The target
compounds 3,6-diaryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-ones (3a-3c) were prepared by compound
2 with substituted alkyl chloride by Williamson reaction. As to 6-benzyl-3-aryl-7H-thiazolo[3,2-
b]-1,2,4-triazin-7-one derivatives as the target compounds, the benzaldehyde and acetylglycine
used as raw materials, followed by Erlenmeyer-Plöchl reaction, condensation reaction, hydrolysis
reaction, condensation reaction, 6-benzyl-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one derivatives
were obtained, and were converted to the target compounds 6-benzyl-3-(hydroxylaryl)-7H-
thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives (5a-5d) using reaction with substituted ꢀ-phenacyl
chlorides. Finally, Williamson reaction were used to yield 6-benzyl-3-aryl-7H-thiazolo[3,2-b]-
1,2,4-triazin-7-ones as target compounds (6a-6e). The ꢀ-catenin translocation capability and the
ALP activation activity were tested, and the glycogen synthase kinase-3 (GSK-3) inhibition was
simulated by molecular docking.
A R T I C L E H I S T O R Y
Received: February 13, 2017
Revised: September 05, 2017
Accepted: September 11, 2017
DOI:
10.2174/1573406413666171002121443
Results: Fourteen 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were synthesized and charac-
terized by mass spectra, proton NMR and infrared spectra, the ꢀ-catenin translocation capability
and the ALP activation activities of the target compounds were tested and calculated. The EC50
value of the ALP activation activity of 6-(4-chlorobenzyl)-3-{4-[(2-dimethylamino)-2-
oxoethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (6b) was 11.283 ꢁM. The molecular
docking results have showed that the target compounds would be GSK-3 inhibitors.
Conclusion: Based on the results of the biological activity test, the target compounds have exhib-
ited the ꢀ-catenin translocation capability and the ALP activation activity.
Keywords: Acetylglycine, alkaline phosphatase, anti-osteoporotic activity, benzaldehyde, ꢀ–Catenin, triazine.
1. INTRODUCTION
report found that the incidence of OP among people aged 50-
60 was 21%, with 60-70 was 58% and with age 70-80 was
100%. Because early clinical manifestations of OP are very
subtle, it has been called “the epidemic disease in silence”
[1].
Osteoporosis (OP) is a common bone disease, most often
diagnosed in postmenopausal women. One investigation
*Address correspondence to these authors at the Key Laboratory of Struc-
ture-based Drug Design & Discovery, Ministry of Education, Shenyang
Pharmaceutical University, Shenyang 110016, P.R. China; E-mails:
zhejin@syphu.edu.cn; chunhu@syphu.edu.cn
At present, the majority of OP treatments are based on
estrogen replacement therapy (ERT), selective estrogen re-
ceptor modulators (SERMs), bisphosphonates, and calci-
1875-6638/18 $58.00+.00
© 2018 Bentham Science Publishers

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Medicinal Chemistry, 2018, Vol. 14, No. 1
Hou et al.
tonin; among them, ERT, SERMs, and calcitonin were fo-
cused at manipulation of the patient’s hormone levels, there-
fore, they were associated with significant adverse effects
such as atrial fibrillation, bone pain, atypical fractures, and
osteosarcoma [2-4]. Novel OP treatments based on molecu-
lar targets, such as the key proteins or nucleic acids with
some particular signaling pathways, such as RANKL, Wnt
and BMP rather than hormones, are therefore urgently
needed.
then collected and crystallized from ethanol to obtain the
target compounds 2a and 2b.
2.1.2. 6-(4-Bromophenyl)-3-(4-hydroxyphenyl)-7H-thiazolo
[3,2-b]-1,2,4-triazin-7-one (2a)
This compound was obtained as a yellow powder, 23%
yield; mp: 292-294°C; ESI-MS (m/z): 397.9 ([M-H]ꢀ), 399.8
([M+2-H]ꢀ); 1H-NMR (300 MHz, DMSO-d6): ꢀ 8.02 (2H, d,
J = 8.52 Hz), 7.69 (2H, d, J = 8.52 Hz), 7.58 (2H, d, J = 8.55
Hz), 7.39 (1H, s), 6.90 (2H, d, J = 8.55 Hz); IR (KBr): ꢂ
3424, 3114, 2445, 1704, 1610, 1589, 1505, 1464, 1384,
1275, 1174, 1076, 1011, 833, 647 cm-1.
Wnt protein is a secreted glycoprotein, consisting of a
frizzled (Fzd) receptor, low-density lipoprotein receptor-
related protein receptor-5/6 (LRP-5/6), disheveled (Dvl) pro-
tein, glycogen synthase kinase-3 (GSK-3), ꢁ–catenin, and
alkaline phosphatase (ALP) in C2C12 cells. In the signaling
pathway, after Wnt is combined with its receptors, Fzd,
LRP-5/6, and Dvl are activated subsequently; then, the GSK-
3 is inhibited by Dvl, which increases ꢁ–catenin transloca-
tion and cellular ALP activation [5]. Following the inhibition
of a GSK-3, ꢁ–catenin is translocated from the adhesion
junctions between the clusters of C2C12 cells to the nucleus
and cytosol, and the ALP activation activity of cell is in-
creased optimally after a 48 h treatment with a GSK-3 in-
hibitor. The untreated cells display very low cellular activity.
The ALP activation activity is a marker for osteoblast differ-
entiation. The ꢁ–catenin translocation and cellular ALP acti-
vation can be measured by laser scanning cytometry [6].
2.1.3. 6-(4-Bromophenyl)-3-(4-hydroxy-3-methoxyphenyl)-
7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (2b)
This compound was obtained as a yellow powder, 17%
yield; mp: 232-234°C; ESI-MS (m/z): 427.9 ([M-H]ꢀ), 429.8
([M+2-H]ꢀ); 1H-NMR (600 MHz, DMSO-d6): ꢀ 8.06 (2H, d,
J = 8.4 Hz), 7.72 (2H, d), 7.46 (1H, s), 7.39 (1H, s), 7.21
(1H, d), 6.94 (1H, d), 3.82 (3H, s); IR (KBr): ꢂ 3423, 3103,
2933, 1664, 1587, 1513, 1469, 1384, 1267, 1180, 1034,
1010, 838, 816, 657, 525 cm-1.
2.1.4. General Procedure for Synthesis of 3,6-diaryl-7H-
thiazolo[3,2-b]-1,2,4-triazin-7-ones (3)
A mixture of 6-(4-Bromophenyl)-3-(4-hydroxy-3-aryl)-
7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (2a or 2b, 10 mmol),
substituted alkyl chlorides (10 mmol), potassium carbonate
(6.9 g, 50 mmol), potassium iodide (0.2 g, 1 mmol), and ace-
tone (25 mL) was added in a round bottom flask, heated and
refluxed for 12-24 h. Then the mixture was poured into a
flask holding cold water (100 mL) and stirred for 12 h. The
solid was collected and recrystallized from absolute ethanol,
to obtain the target compounds 3a-3c.
In this work, based on virtual screening, scaffold hopping
and structural optimization, a series of 7H-thiazolo[3,2-b]-
1,2,4-triazin-7-one derivatives was synthesized and evalu-
ated for the ꢁ-catenin translocation capability and the ALP
activation activity, the glycogen synthase kinase-3 (GSK-3)
inhibition was simulated by molecular docking.
2. MATERIAL AND METHODS
2.1. Chemistry
2.1.5. 6-(4-Bromophenyl)-3-{4-[(2-benzylamino)-2-oxo-eth-
oxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (3a)
All reagents and solvents were purchased from common
commercial suppliers and were used without further purifica-
tion. Melting points were obtained using a Koffler hot-plate
apparatus. Mass spectra (MS) were determined on an elec-
trospray (ESI) ion source in a Waters spectrometer at 3.5 kV
This compound was obtained as a yellow powder, 44%
yield; mp: 220-222°C; ESI-MS (m/z): 547.1 ([M+H]+),
569.1 ([M+Na]+), 571.1 ([M+2+Na]+), 585.1 ([M+K]+),
587.1 ([M+2+K]+); 1H-NMR (600 MHz, DMSO-d6): ꢀ 8.72
(1H, t), 8.02 (2H, d), 7.73 (2H, d), 7.69 (2H, d), 7.49 (1H, s),
7.22~7.31 (5H, m), 7.14 (2H, d), 4.66 (2H, s), 4.36 (2H, s);
IR (KBr): ꢂ 3424, 3268, 3074, 2922, 1660, 1631, 1534,
1505, 1464, 1384, 1235, 1178, 1078, 1009, 834, 738, 552,
527 cm-1.
1
spray voltage, and acetonitrile was used as the solvent. H-
NMR spectra were determined in DMSO-d6 or CDCl3 on a
Bruker spectrometer operating at 300 MHz or 600 MHz. The
IR spectra were obtained using a Bruker AFS55 spectrome-
ter.
The target compounds 6-aryl-3-thioxo-3,4-dihydro-1,2,4-
triazin-5(2H)-one (1) and 6-arylmethyl-3,4-dihydro-3-
thioxo-1,2,4-triazin-5(2H)-one (4) have been reported previ-
ously [7-10]. The synthesis of compounds 2, 3, 5, 6 was as
follows:
2.1.6. 6-(4-Bromophenyl)-3-{4-[2-(4-chlorobenzylamino)-2-
oxoethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one
(3b)
This compound was obtained as a yellow powder, 66%
yield; mp: 269-271°C; ESI-MS (m/z): 567.1 ([M+H]+),
589.0 ([M+Na]+), 591.0 ([M+2+Na]+), 605.0 ([M+K]+),
607.0 ([M+2+K]+); 1H-NMR (600 MHz, DMSO-d6): ꢀ 11.08
(1H, s), 8.02 (2H, d, J = 7.8 Hz), 7.68~7.73 (6H, m), 7.50
(1H, s), 7.38 (2H, d, J = 7.8 Hz), 7.16 (2H, d, J = 7.8 Hz),
4.87 (2H, s); IR (KBr): ꢂ 3406, 3075, 1694, 1584, 1538,
1492, 1466, 1401, 1385, 1248, 1179, 1079, 1011, 829, 649,
554, 523 cm-1.
2.1.1. General Procedure for Synthesis of 3,6-diaryl-7H-
thiazolo[3,2-b]-1,2,4-triazin-7-ones (2)
A mixture of 6-aryl-3-thioxo-3,4-dihydro-1,2,4-triazin-
5(2H)-one (10 mmol) and substituted phenacyl chloride (10
mmol) was dissolved in glacial acetic acid (20 mL) and re-
fluxed for 12-24 h. When the solution was cooled down to
room temperature, the solid was precipitated and filtered and

Synthesis, ꢀ-catenin Translocation Capability and ALP Activation Activity
Medicinal Chemistry, 2018, Vol. 14, No. 1 69
1475, 1405, 1351, 1268, 1199, 1126, 1052, 897, 867, 819,
738, 702 cm-1.
2.1.7. 6-(4-Bromophenyl)-3-{3-methoxy-4-[2-(4-chloroben-
zylamino)-2-oxoethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-
triazin-7-one (3c)
2.1.13. General Procedure for Synthesis of 6-arylmethyl-3-
aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7- ones (6)
This compound was obtained as a yellow powder, 77%
yield; mp: 226-229°C; ESI-MS (m/z): 594.8 ([M-H]ꢀ), 596.8
([M+2-H]ꢀ); 1H-NMR (600 MHz, DMSO-d6): ꢀ 10.48 (1H,
s), 8.05 (2H, d, J = 8.4 Hz), 7.70 (2H, d, J = 8.4 Hz), 7.68
(2H, d, J = 9.0 Hz), 7.52 (1H, d), 7.49 (1H, s), 7.38 (2H, d, J
= 8.4 Hz), 7.31 (1H, s), 7.09 (1H, d), 4.82 (2H, s), 3.88 (3H,
s); IR (KBr): ꢁ 3432, 2928, 1642, 1541, 1492, 1468, 1401,
1258, 1145, 1081, 1033, 1012, 833, 527, 506 cm-1.
6-Arylmethyl-3-(4-hydroxyaryl)-7H-thiazolo[3,2-b]-1,2,
4-triazin-7-one (5a-5d, 10 mmol) was dissolved in acetone
(20 mL). Potassium carbonate (2.0 g, 14.5 mmol) and substi-
tuted alkyl chloride (10 mmol) were added, and the mixture
was refluxed for 8-24 h, until the TLC indicated the comple-
tion of the reaction. The mixture was filtered to obtain crude
product which was evaporated to remove solvents and was
collected and recrystallized from ethanol, to give the target
compound 6.
2.1.8. General Procedure for Synthesis of 6-arylmethyl-3-
aryl-7H-thiazolo[3,2-b]-1,2,4-triazin-7- ones (5)
A mixture of 6-arylmethyl-3,4-dihydro-3-thioxo-1,2,4-
triazin-5(2H)-one (4, 10 mmol), substituted phenacyl chlo-
ride (10 mmol) and AcONa 2.0 g (24.4 mmol), was dis-
solved in glacial acetic acid 20 mL and refluxed for 10-24 h.
The progress of the reaction was monitored by TLC, the re-
action liquid was cooled down to room temperature, which
was filtered and crystallized from ethanol to obtain the target
compounds (5).
2.1.14. 6-Benzyl-3-{4-[2-(1-piperidinyl)ethoxy]phenyl}-7H-
thiazolo[3,2-b]-1,2,4-triazin-7-one (6a)
This compound was obtained as a pale yellow powder,
52% yield; mp: 185-187°C; ESI-MS (m/z): 447.0 ([M+H]+),
1
893.0 ([2M+H]+), 915.9 ([2M+Na]+); H-NMR (300 MHz,
CDCl3): ꢀ 7.46 (2H, d, J = 8.8 Hz), 7.25-7.36 (5H, m), 6.92
(2H, d, J = 8.8 Hz), 6.72 (1H, s), 4.18 (2H, t), 4.12 (2H, s),
2.83 (2H, t), 2.56 (4H, t), 1.65 (4H, m), 1.48 (2H, m); IR
(KBr): ꢁ 3109, 2931, 2851, 1634, 1579, 1507, 1480, 1416,
1386, 1356, 1293, 1253, 1173, 1036, 933, 824, 767 cm-1.
2.1.9.
6-Benzyl-3-(4-hydroxyphenyl)-7H-thiazolo[3,2-b]-
1,2,4-triazin-7-one (5a)
This compound was obtained as a pale yellow crystal,
85% yield; mp: 245-247°C; ESI-MS (m/z): 335.9 ([M+H]+);
1H-NMR (600 MHz, DMSO-d6): ꢀ 9.92 (1H, s), 7.46 (2H, d,
J = 8.4 Hz), 7.33 (1H, s), 7.23-7.30 (5H, m), 6.78 (2H, d, J =
8.4 Hz), 3.98 (2H, s); IR (KBr): ꢁ 3229, 3035, 2941, 1604,
1557, 1511, 1472, 1440, 1386, 1345, 1299, 1275, 1229,
1206, 1177, 1127, 1071, 842, 776, 757, 749, 722, 702 cm-1.
2.1.15. 6-(4-Chlorobenzyl)-3-{4-[(2-dimethylamino)-2-oxo-
ethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (6b)
This compound was obtained as a white powder, 42%
1
yield; mp: 192-194°C; ESI-MS (m/z): 454.9 ([M+H]+); H-
NMR (600 MHz, DMSO-d6): ꢀ 7.51 (2H, d, J = 8.4 Hz),
7.41 (1H, s), 7.37 (2H, d, J = 8.4 Hz), 7.32 (2H, d, J = 8.4
Hz), 6.92 (2H, d, J = 8.4 Hz), 4.89 (2H, s), 3.96 (2H, s), 3.02
(3H, s), 2.86 (3H, t); IR (KBr): ꢁ 3124, 2921, 1655, 1608,
1579, 1491, 1417, 1384, 1357, 1309, 1297, 1271, 1249,
1183, 1151, 1106, 1062, 1014, 950, 883, 815, 788, 767, 743,
729 cm-1.
2.1.10. 6-(4-Chlorobenzyl)-3-(4-hydroxyphenyl)-7H-thia-
zolo[3,2-b]-1,2,4-triazin-7-one (5b)
This compound was obtained as a white powder, 65%
yield; mp: 219-221°C; ESI-MS (m/z): 369.9 ([M+H]+),
1
762.8 ([2M+Na]+); H-NMR (300 MHz, DMSO-d6): ꢀ 9.93
2.1.16. 6-(4-Methoxybenzyl)-3-{4-[2-(1-piperidinyl)ethoxy]
phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (6c)
(1H, s), 7.43 (2H, d, J = 8.6 Hz), 7.30-7.37 (5H, m), 6.78
(2H, d, J = 8.6 Hz), 3.98 (2H, s); IR (KBr): ꢁ 3425, 1647,
1611, 1509, 1480, 1384, 1277, 1236, 1179, 1092, 1050,
1016, 837, 808, 751 cm -1.
This compound was obtained as a pale yellow powder,
37% yield; mp: 171-173°C; ESI-MS (m/z): 477.7 ([M+H]+);
1H-NMR (300 MHz, CDCl3): ꢀ 7.47 (2H, d, J = 8.7 Hz),
7.27 (2H, d, J = 8.4Hz), 6.94 (2H, d, J = 8.7 Hz), 6.83 (2H,
d, J = 8.4 Hz), 6.72 (1H, s), 4.20 (2H, t), 4.05 (2H, s), 3.79
(3H, s), 2.86 (2H, t), 2.59 (4H, s), 1.64 (4H, s), 1.49 (2H, s);
IR (KBr): ꢁ 3020, 2933, 1632, 1580, 1512, 1479, 1385,
1355, 1300, 1252, 1180, 1118, 1035, 820, 760 cm-1.
2.1.11. 6-(4-Methoxybenzyl)-3-(4-hydroxyphenyl)-7H-thia-
zolo[3,2-b]-1,2,4-triazin-7-one (5c)
This compound was obtained as a pale yellow powder,
64% yield; mp: 188-189°C; ESI-MS (m/z): 366.0 ([M+H]+),
1
752.8 ([2M+Na]+); H-NMR (600 MHz, DMSO-d6): ꢀ 10.0
(1H, s), 7.48 (2H, d, J = 8.4 Hz), 7.33 (1H, s), 7.21 (2H, d, J
= 8.4 Hz), 6.87 (2H, d, J = 8.4 Hz), 6.80 (2H, d, J = 8.4 Hz),
3.91 (2H, s), 3.76 (3H, s); IR (KBr): ꢁ 3119, 1610, 1511,
1476, 1385, 1279, 1247, 1176, 1123, 1029, 838, 752 cm-1.
2.1.17. 6-Benzyl-3-{3-methyl-4-[2-(1-piperidinyl)ethoxy]ph-
enyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (6d)
This compound was obtained as a white powder, 43%
1
yield; mp: 168-169°C; ESI-MS (m/z): 461.0 ([M+H]+); H-
2.1.12. 6-Benzyl-3-(3-methyl-4-hydroxyphenyl)-7H-thiazolo
[3,2-b]-1,2,4-triazin-7-one (5d)
NMR (600 MHz, DMSO-d6): ꢀ 7.43 (1H, d), 7.38 (2H, s),
7.23~7.30 (5H, m), 6.98 (1H, d), 4.13 (2H, t), 3.88 (2H, s),
2.70 (2H, t), 2.46 (4H, t), 2.13 (3H, s), 1.50 (4H, t), 1.39
(2H, s); IR (KBr): ꢁ 3109, 2931, 2785, 1634, 1575, 1478,
1385, 1359, 1305, 1257, 1134, 1037, 957, 883, 859, 815,
799, 767, 750, 708 cm-1.
This compound was obtained as a pale yellow powder,
73% yield; mp: 224-225°C; ESI-MS (m/z): 350.0 ([M+H]+),
1
720.9 ([2M+Na]+); H-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); IR (KBr): ꢁ 3226, 1624, 1604, 1559,

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Hou et al.
confirmed as a GSK-3 inhibitor [16]. The complex 4IQ6 was
used for taking molecular docking, the interactions of GSK-3
and 6b, GSK-3 and 7, and GSK-3 and 8 were analyzed by
using Molegro Virtual Docker (MVD) software, and the
docking results were compared. The parameters of MVD
were all default values.
2.1.18. 6-Benzyl-3-{3-methyl-4-[2-(4-morpholinyl)ethoxy]
phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (6e)
This compound was obtained as a white powder, 38%
yield; mp: 174-176°C; ESI-MS (m/z): 463.2 ([M+H]+),
1
925.3 ([2M+H]+); H-NMR (300 MHz, CDCl3): ꢀ 7.24~7.39
(7H, m), 6.83 (1H, d), 6.70 (1H, s), 4.20 (2H, t), 4.13 (2H, s),
3.77 (4H, t), 2.90 (2H, t), 2.65 (4H, t), 2.24 (3H, s); IR
(KBr): ꢂ 3088, 2949, 2923, 2856, 2799, 1626, 1573, 1560,
1472, 1386, 1357, 1303, 1261, 1213, 1170, 1137, 1116,
1069, 1041, 960, 939, 900, 859, 802, 703 cm-1.
3. RESULTS AND DISCUSSION
3.1. Chemistry
The synthesis of the target compounds 6-aryl-3-thioxo-
3,4-dihydro-1,2,4-triazin-5(2H)-one (1) and 6- arylmethyl-
3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one (4) has been
reported [7-10].
2.2. Biological Activity
The target compounds were evaluated for the ꢁ-catenin
translocation capability and the ALP activation activity using
the Eli Lilly Open Innovation Drug Discovery Platform. The
Wnt3A conditioned media were prepared as described [11].
C2C12 cells seeded in 384-well plates were treated with
Wnt3A, bone morphogenetic protein 4, and the target com-
pounds at the indicated concentration for either 24 h or 48 h
respectively, before measuring ꢁ-catenin located in the cell
nuclei or cell-associated ALP activation activity [6]. The ꢁ-
catenin translocation capability (% stimulation) of the target
compounds at 10 ꢂM concentration was tested and calcu-
lated. Then, for the target compounds whose ꢁ-catenin trans-
location capability (% stimulation) values were higher than
60%, the EC50 values of the ALP activation activities were
tested and calculated.
The general synthesis procedure is described as follows:
The target compounds 3,6-diaryl-7H-thiazolo[3,2-b]-1,2,4-
triazin-7-ones (2) were prepared by 1 reacted with substi-
tuted phenacyl chlorides (2-chloro-1-(4-hydroxyphenyl)
ethan-1-one or 2-chloro-1-(4-hydroxy-3- methoxyphenyl)
ethan-1-one) in acetic acid to give 17% and 23% yield, re-
spectively. The target compounds 3,6-diaryl-7H-thia-
zolo[3,2-b]-1,2,4-triazin-7-ones (3) were obtained by the
classical Williamson reaction of 2 reacted with substituted
alkyl chlorides, potassium carbonate, and potassium iodide
in acetone with 44%-77% yield. The synthetic route is
shown in Scheme 1.
The
target
compounds
6-arylmethyl-3-aryl-7H-
thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives (5) were ob-
tained by 4 reacted with different substituted phenacyl chlo-
rides in acetic acid and sodium acetate to give the corre-
sponding target compounds 5 in 64%-85% yield. The target
compounds 6-arylmethyl-3-aryl-7H-thiazolo[3,2-b]-1,2,4-tri-
azin-7-ones (6) were prepared by the Williamson reaction of
5 reacted with substituted alkyl chloride in acetone and po-
tassium carbonate with 37%-55% yield. The synthetic route
is shown in Scheme 2.
2.3. Molecular Docking
GSK-3 is a multifunctional serine-threonine kinase,
which plays an important role in glycogen metabolism, Wnt
and hedgehog signal transduction pathways. Some crystal
structure complexes which consist of GSK-3 and its small
molecule inhibitor have been reported in Research Collabo-
ratory for Structural Bioinformatics (RCSB) protein data
bank. The PDB ID of the complexes was 4IQ6 [12], 1Q3W
[13], 4ACD [14], 4B7T [15], and so on.
The chemical structures of all target compounds were
1
fully characterized by mass spectra (MS), H-NMR, and in-
frared spectra data.
Complex 4IQ6 consists of GSK-3 and the small molecule
GSK-3 inhibitor 6-chloro-N-cyclohexyl-4-(1H-pyrrolo[2,3-
b]pyridin-3-yl)pyridin-2-amine (7) [12]. Compound 8 was
R2
O
R1
R2
O
Br
Cl
R1
Br
KMnO4
NaOH
NH2NHCSNH2
ethanol
HCl
COOH
N
N
N
NH
N
Br
Br
AcOH, AcONa
O
N
H
S
S
O
1
2
R2
OH
R1
R1
Br
Br
O
O
Substituted alkyl chloride
N
N
N
NH
N
N
O
Cl
KI, K2CO3, acetone
3a: R1 = H, R2
=
S
S
O
O
N
O
NH
Cl
3
2a-2b
2a: R1 = H, R2 = OH
2b: R1 = OCH3, R2 = OH
3b: R1 = H, R2 =
O
NH
1
2
O
3c: R = OCH3, R =
Scheme 1. The synthetic route of 2a-2b and 3a-3c.

Synthesis, ꢀ-catenin Translocation Capability and ALP Activation Activity
Medicinal Chemistry, 2018, Vol. 14, No. 1 71
O
O
O
H
N
acetone, H2O
refluxed
Ac2O, AcONa
HCl
OH
R3
CHO
O
OH
HN
N
refluxed
refluxed
R3
R3
O
O
R5
O
O
R4
R4
R5
Cl
N
OH
NH
NH2NHCSNH2
ethanol, refluxed
N
N
O
R3
R3
O
N
H
S
AcOH, AcONa, refluxed
R3
O
N
S
5
4
OH
R5
N
R4
O
R4
6a: R3 = H, R4 = H, R5 =
6b: R3 = Cl, R4 = H, R5 =
O
N
N
Substituted alkyl chloride
KI, K2CO3, acetone, refluxed
N
N
N
N
O
R3
O
S
N
R3
O
N
S
O
6c: R3 = OCH3, R4 = H, R5 =
6d: R3 = H, R4 = CH3, R5 =
6e: R3 = H, R4 = CH3, R5 =
5a-5d
6
N
5a: R3 = H, R4 = H, R5 = OH
5b: R3 = Cl, R4 = H, R5 = OH
5c: R3 = OCH3, R4 = H, R5 = OH
5d: R3 = H, R4 = CH3, R5 = OH
O
N
O
O
Scheme 2. The synthetic route of 5a-5d and 6a-6e.
Table 1. The ꢀ-catenin translocation capability of the target compounds (3a-3c, 5b, 6a-6e).
Compound No.
Stimulation, %
SD
Compound No.
Stimulation, %
SD
3a
3b
3c
5b
6a
12.94
7.43
0.135
0.0831
0.0357
0.372
6b
6c
6d
6e
-
74.15
3.39
9.29
6.31
-
0.296
0.0598
0.0865
0.0527
-
2.72
64.16
63.86
0.323
Table 2. EC50 values of the ALP activation activity of the target compounds (5b, 6a, 6b).
Compound No.
5b
EC50, ꢀM
Compound No.
6a
EC50, ꢀM
Compound No.
6b
EC50, ꢀM
>20
14.751
11.283
3.2. Biological Activity
the target compound 6b, which had a stronger ALP activa-
tion activity than others, the EC50 value was 11.283 ꢃM.
The ꢀ-catenin translocation capability (% stimulation) of
the tested compounds at the indicated concentration (10 ꢃM)
was tested and calculated.
3.3. Molecular Docking
The interactions of GSK-3 and the target compound 6b,
GSK-3 and the active ligand 7, GSK-3 and its inhibitor 8,
were analyzed by MVD software and compared as shown in
Fig. (2) and Table 3.
The experimental results are listed in Table 1.
The target compounds 5b, 6a, 6b were chosen for further
screening. The compounds at the indicated concentration
showed higher ꢀ-catenin translocation capability. The EC50
values of the ALP activation activities in C2C12 cells were
tested and calculated. The experimental results were listed in
Table 2 and Fig. (1).
The results of the molecular docking showed that the in-
teraction at the active sites and the interaction modes of
GSK-3 and 6b, GSK-3 and 7, and GSK-3 and 8 were similar.
Hydrogen bond interactions were found between 6b and
GSK-3, 7 and GSK-3, 8 and GSK-3 at amino acid residues
Tyr140 and Ser147.
From the biological data of Table 2 and (Fig. 1), the EC50
values of the three target compounds were calculated. For

72 Medicinal Chemistry, 2018, Vol. 14, No. 1
Hou et al.
Wnt3a_C2C12 Osteogen AlkPhos
179978563
Wnt3a_C2C12 Osteogen AlkPhos
183380486
EC50 EC50 = 14.751 uM
Wnt3a_C2C12 Osteogen AlkPhos
180779252
EC50 > 20 uM
EC50
EC50 = 11.283 uM
120
100
80
60
40
20
0
120
100
80
60
40
20
0
120
100
80
60
40
20
0
-20
0.001
-20
0.001
-20
0.001
0.01
0.1
1
10
100
0.01
0.1
1
10
100
0.01
0.1
1
10
100
Conc. (uM)
51153506, 2011-06-09
Conc. (uM)
51153512, 2011-06-09
Conc. (uM)
51153503, 2011-06-09
5b
6a
6b
Fig. (1). EC50 values of the ALP activation activity of the target compounds (5b, 6a, 6b).
SER
SER
ARG
A:147
A:147
A: 148
CI
CI
+
CI
ARG
CI
N
Pi
H
N
SER
A:144
+
O
N
A:147
N
N
N
O
N
N
N
CI
N
Pi
H
N
N
Pi
Pi
H
N
N
H
Pi
O
N
+
H
ARG
Pi
A:148
N
TYR
A:140
TYR
A:140
S
+
GLN
A:185
ARG
A:144
TYR
A:140
Fig. (2). The docking model of 6b (left), 7 (middle), and 8 (right) at the active sites of GSK-3.
Table 3. The interaction active sites of GSK-3 and 6b, GSK-3 and 7, GSK-3 and 8.
Amino Acid Residues
No.
Amino Acid Residues
(Other Interactions Between Molecule and GSK-3)
(Hydrogen Bonds Between Molecule and GSK-3)
6b
7
Tyr140, Ser147
Tyr140, Ser147, Gln185
Tyr140
Arg144 (charge), Arg148(charge)
——
8
Arg144 (charge), Arg148(charge)
Ala143
Gln185
Tyr140
Arg144
Ser147
Arg148
Arg220
Fig. (3). The 3D active conformations of compounds 6b (red), 7 (violet) and 8 (green) at the active sites of GSK-3.
The similarity of interaction active sites was further
proved and the three-dimensional active conformations of
compound 6b, 7 and 8 at the active site are shown in Fig. (3).
Comparison of the three-dimensional active conforma-
tions of the active molecule 7, the GSK-3 inhibitor 8 and the
target compound 6b at the active site of GSK-3 showed that

Synthesis, ꢀ-catenin Translocation Capability and ALP Activation Activity
Medicinal Chemistry, 2018, Vol. 14, No. 1 73
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Not applicable.
[10]
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[12]
HUMAN AND ANIMAL RIGHTS
No Animals/Humans were used for studies that are base
of this research.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
[13]
[14]
ACKNOWLEDGEMENTS
This work was supported by the National Science Foun-
dation of China (NFSC) for the grant No. 21072130; the
Natural Science Foundation of Liaoning Province, China for
the grant No. 201602695 and the Scientific Research Foun-
dation of Department of Education of Liaoning Province,
China for the grant No. L2015517. The authors are grateful
for the biological activity screening supporting data of the
Eli Lilly Open Innovation Drug Discovery Platform. The
authors would like to thank Molegro ApS for kindly provid-
ing a free evaluation copy of their software package.
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