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

Article abstract of DOI:10.1111/cbdd.12362

New dual binding site acetylcholinesterase (AChE) inhibitors have been designed and synthesized as a new drug candidate for the treatment of Alzheimer's disease (AD) through the binding to both catalytic and peripheral sites of the enzyme. Therefore, a se

Full text of DOI:10.1111/cbdd.12362

Received Date : 25-Feb-2014
Revised Date : 02-Apr-2014
Accepted Date : 30-Apr-2014
Article type
: Research Article
Design, synthesis, and evaluation of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one
derivatives as dual binding site acetylcholinesterase inhibitors
Sijie Liu1, Ruofeng Shang2*, Lanxiang Shi1, Ran Zhou1, Jinyu He1, David Chi-Cheong Wan3
1College of Chemical Engineering, Shijiazhuang University, Shijiazhuang 050035, PR China
2Key Laboratory of New Animal Drug Project of Gansu Province; Key Laboratory of Veterinary Pharmaceutical
Development, Ministry of Agriculture, P. R. China; Lanzhou Institute of Husbandry and Pharmaceutical
Sciences of CAAS, Lanzhou 730050, PR China
3Department of Biochemistry, The Chinese University of Hong Kong, Hong Kong SAR, PR China
*Corresponding author: Tel.: +86 931 2115286; fax: +86 931 2115951. E-mail address:
shangrf1974@163.com
Abstract: New dual binding site acetylcholinesterase (AChE) inhibitors have been designed and synthesized
as a new drug candidate for the treatment of Alzheimer’s disease (AD) through the binding to both catalytic
and peripheral sites of the enzyme. Therefore, a series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives
This article has been accepted for publication and undergone full peer review but has not been through
the copyediting, typesetting, pagination and proofreading process which may lead to differences
between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi:
10.1111/cbdd.12362
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6a-j were synthesized and investigated for their ability to inhibit the activity of human AChE (hAChE) in
comparison with huperzine-A. All the compounds were found to inhibit AChE activity, especially compounds
6c and 6i with the inhibition value of 76.10 and 77.82%, respectively. The molecular docking study indicated
that they were nicely accommodated by AChE. The molecular docking study revealed that 6c and 6i
possessed a more optimal binding conformation than 6a and can perfectly fit into the active and peripheral
site of hAChE, and consequently exhibited highly improved inhibitor potency to hAChE.
Keywords: acetylcholinesterase; synthesis; inhibitors; 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives;
docking; Alzheimer’s disease
Introduction
Alzheimer’s disease (AD) is described by Dr. Alois Alzheimer in 1907 as a neurodegenerative disorder. It
affects over 20 million individuals worldwide and this number will substantially increase in the future along
with the increase of the number of elderly in the population [1]. AD leads to a progressive decline of the
cognitive function, executive function losses, memory deficits, and eventually to incapacitating dementia
before death [2, 3]. The proven and currently accepted therapeutic approach for the treatment of AD
involves the inhibition of the enzyme acetylcholinesterase (AChE) which is responsible for the hydrolysis of
acetylcholine (ACh), thereby raising the levels of ACh in the synaptic cleft [4]. Five cholinesterase inhibitors
tacrine, donepezil, rivastigmine, huperzine-A and galantamine (Figure 1) are approved by the US Food and
Drug Administration (FDA) and are currently in the market [5].
X-ray structures of AChE co-crystallized with various ligands provided insights into the essential structural
elements and motifs central to its catalytic mechanism and mode of ACh processing. One of the striking
structural features of the Torpedo californica AChE (TcAChE) from the X-ray analysis is the presence of a
narrow, long, hydrophobic gorge which is approximately 20 Å deep [6]. The catalytic active site (CAS) is the
binding site of classical AChE inhibitors, such as tacrine and huperzine A, which has been studied thoroughly.
The CAS comprising Ser203, Glu334 and His447 interacts with the cationic substrates, for example ACh. The
esteratic locus of the aromatic gorge also interacts with hydrophobic substrates and ligands. Interactions
between quaternary ammonium functionality of many ligands and the side chains of Trp86, Glu202, and
Tyr337, and the phenolic oxygen of Tyr133 are found in the anionic binding site near the CAS [7].
Additionally, there is a peripheral anionic site (PAS) composed of Tyr72, Asp74, Tyr124 and Trp286 which
located at the gorge entrance. But the function of the PAS has not been elucidated clearly as yet. Recent
studies have demonstrated that the PAS might accelerate the aggregation and deposition of β-amyloid
peptide which are considered as another cause of AD [8]. Therefore, the ideal AChE inhibitors should bind to
the CAS and PAS simultaneously and disrupt the interactions between the enzyme and the β-amyloid
peptide, and then slow down the progression of the disease.
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In an earlier report, we have presented the preparation and evaluation of some new
7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives as AChE inhibitors which interacted with the PAS and the
CAS of AchE [9-11]. Most of these compounds proved to be potent AChE inhibitors in vitro, among which
compounds bearing two substituents on the aryl ring at C3 position of parent nucleus ring showed the most
activity. Following these reasons and in pursuit of our previous study a series of new
7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were designed, synthesized and evaluated for AChE
inhibitory activities. The final compounds have been tested for their ability to inhibit AChE using Ellman’s
method [12].
Materials and Methods
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 a Perkin-Elmer 298 spectrometer (Perkin-Elmer, Norwalk, CT, USA).
4-[(4-Methoxyphenyl)methylene]-2-methyl-5(4H)-oxazolone (1),
2-Acetamido-3-(4-methoxyphenyl)-2-propenoic acid (2), 4-Methoxyphenylpyuvic acid (3),
6-(4-Methoxybenzyl)-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one (4) and
6-(4-methoxybenzyl)-3-hydroxylphenyl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-ones (5) were prepared by
previously reported procedures[9].
General procedure for synthesis of 6-(4-methoxybenzyl)-3-aryl-7H-thiazolo[3,2-b]-1,2,4 -triazin-7-ones (6)
6-(4-methoxybenzyl)-3-hydroxylphenyl-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (5a-b) (10 mmol) was solved in
acetone (20mL), K2CO3 (2g) and alkyl chloride (10 mmol) were added, and refluxed for 8-24h until the TLC
assay indicated that the reaction was completed. The mixture was filtered, and evaporated to remove
solvents. The residues were collected and recrystallized from ethanol, to give the target compounds 6a-j.
6-(4-methoxybenzyl)-3-{4-[2-(dimethylamino)ethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (6a):
white crystal, 41.2% yield; mp: 176-178 ; 1H-NMR (300 MHz, CDCl3): δ 7.47 (2H, d, J = 8.7Hz), 7.27 (2H, d, J =
9.0Hz), 6.97 (2H, d, J = 9.0Hz), 6.83 (2H, d, J = 8.7Hz), 6.71 (1H, s), 4.14 (2H, t), 4.05 (2H, s), 3.79 (3H, s), 2.80
(2H, t), 2.39 (6H, s); ESI-MS (m/z): 437.0 (M+H)+; IR (KBr): υ 2930, 1624, 1477, 1384, 1251, 1178, 1034, 810
cm-1.
6-(4-methoxybenzyl)-3-{4-[2-(1-piperidinyl)ethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one (6b):
yellow crystal, 36.7% yield; mp: 171-173 ; 1H-NMR (300 MHz, CDCl3): δ 7.47 (2H, d, J = 8.7Hz), 7.27 (2H, d, J =
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8.4Hz), 6.94 (2H, d, J = 8.7Hz), 6.83 (2H, d, J = 8.4Hz), 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); ESI-MS (m/z): 477.7 (M+H)+; IR (KBr): υ 3020, 2933, 1632, 1580,
1512, 1479, 1385, 1355, 1300, 1252, 1180, 1118, 1035, 760 cm-1.
6-(4-methoxybenzyl)-3-{3-methyl-4-[2-(dimethylamino)ethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-o
ne (6c): yellow crystal, 32.2% yield; mp: 157-158 ; 1H-NMR (600 MHz, CDCl3): δ 7.35 (1H, d), 7.32 (1H, s),
7.28 (2H, d, J = 8.4Hz), 6.84 (1H, d), 6.83 (2H, d, J = 8.4Hz), 6.68 (1H, s), 4.17 (2H, t), 4.05 (2H, s), 3.78 (3H, s),
2.84 (2H, t), 2.40 (6H, s), 2.25 (3H, s); ESI-MS (m/z): 451.0 (M+H)+; IR (KBr): υ 2939, 1630, 1487, 1384, 1301,
1247, 1177, 1135, 1034, 800 cm-1.
6-(4-methoxybenzyl)-3-{3-methyl-4-[2-(1-piperidinyl)ethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-one
(6d): yellow crystal, 45.9% yield; mp: 165-166 ; 1H-NMR (600 MHz, CDCl3): δ 7.36 (1H, d), 7.32 (1H, s), 7.29
(2H, d, J = 8.4Hz), 6.84 (1H, d), 6.83 (2H, d, J = 8.4Hz), 6.68 (1H, s), 4.21 (2H, s), 4.06 (2H, s), 3.80 (3H, s), 2.90
(2H, s), 2.59 (4H, s), 2.24 (3H, s), 1.65 (4H, s), 1.48 (2H, s); 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.
6-(4-methoxybenzyl)-3-{3-methyl-4-[2-(4-morpholinyl)ethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-on
e (6e): yellow crystal,41.6% yield; mp: 162-164 ; 1H-NMR (300 MHz, CDCl3): δ 7.28~7.35 (4H, m), 6.85 (3H,
m), 6.70 (1H, s), 4.21 (2H, t), 4.07 (2H, s), 3.80 (3H, s), 3.77 (4H, m), 2.90 (2H, t), 2.66 (4H, m), 2.26 (3H, s);
ESI-MS (m/z): 493.0 (M+H)+, 985.9 (2M+H)+; IR (KBr): υ 2932, 1636, 1489, 1384, 1248, 1118 cm-1.
6-(4-methoxybenzyl)-3-{3-methyl-4-[2-(diethylamino)ethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin-7-on
e (6f): yellow crystal, 35.5% yield; mp: 158-159 ; 1H-NMR (300 MHz, CDCl3): δ 7.29~7.37 (4H, m), 6.84 (3H, t),
6.67 (1H, s), 4.12 (2H, t), 4.05 (2H, s), 3.78 (3H, s), 2.96 (2H, t), 2.69 (4H, m), 2.24 (3H, s), 1.11 (6H, m); ESI-MS
(m/z): 479.1 (M+H)+; IR (KBr): υ 2967, 1628, 1487, 1384, 1302, 1259, 1176, 1136, 1038, 800 cm-1.
6-(4-methoxybenzyl)-3-{3-methyl-4-[2-(1-piperidinyl)-2-oxoethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazin
-7-one (6g): yellow crystal, 32.7% yield; mp: 133-134 ; 1H-NMR (300 MHz, CDCl3): δ 7.36 (1H, d), 7.32 (1H, s),
7.29 (2H, d, J = 8.4Hz), 6.84 (1H, d), 6.83 (2H, d, J = 8.4Hz), 6.68 (1H, s), 4.76 (2H, s), 4.05 (2H, s), 3.79 (3H, s),
3.59 (2H, s), 2.54 (2H, s), 2.29 (3H, s), 1.65 (4H, s), 1.48 (2H, s); ESI-MS (m/z): 505.0 (M+H)+; IR (KBr): υ 2935,
2856, 1637, 1513, 1481, 1384, 1302, 1248, 1177, 1135, 1032, 807, 758 cm-1.
6-(4-methoxybenzyl)-3-{3-methyl-4-[2-(4-morpholinyl)-2-oxoethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazi
n7-one (6h): yellow crystal,33.6% yield; mp: 152-154 ; 1H-NMR (300 MHz, CDCl3): δ 7.37 (2H, d), 7.29 (2H, d,
J = 8.5Hz), 6.90 (1H, d), 6.84 (2H, d, J = 8.5Hz), 6.71 (1H, s), 4.81 (2H, s), 4.10 (2H, s), 3.80 (3H, s), 3.68 (8H,
m), 2.29 (3H, s); ESI-MS (m/z): 507.0 (M+H)+; IR (KBr): υ 3096, 2963, 2853, 1632, 1570, 1513, 1482, 1384,
1359, 1302, 1248, 1177, 1135, 1116, 1068, 1031, 971, 819, 750 cm-1.
6-(4-methoxybenzyl)-3-{3-methyl-4-[(2-diethylamino)-2-oxoethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-triazi
n-7-one (6i): yellow crystal, 37.6% yield; mp: 132-134 ; 1H-NMR (300 MHz, CDCl3): δ 7.37 (2H, d), 7.29 (2H,
d), 6.86 (3H, t), 6.70 (1H, s), 4.80 (2H, s), 4.06 (2H, s), 3.80 (3H, s), 3.46 (4H, m), 2.31 (3H, s), 1.27 (3H, t), 1.19
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(3H, t); ESI-MS (m/z): 493.0 (M+H)+; IR (KBr): υ 3067, 2933, 1634, 1567, 1513, 1481, 1383, 160, 1302, 1249,
1177, 1134, 1070, 1033, 867, 800, 757 cm-1.
6-(4-methoxybenzyl)-3-{3-methyl-4-[2-(dimethylamino)-2-oxoethoxy]phenyl}-7H-thiazolo[3,2-b]-1,2,4-tria
zin-7-one (6j): yellow crystal, 34.4% yield; mp: 126-127 ; 1H-NMR (300 MHz, CDCl3): δ 7.26~7.34 (4H, t), 6.85
(3H, d), 6.70 (1H, s), 4.80 (2H, s), 4.05 (2H, s), 3.80 (3H, s), 3.14 (3H, s), 3.02 (3H, s), 2.37 (3H, s); ESI-MS (m/z):
465.0 (M+H)+; IR (KBr): υ 3076, 2928, 1637, 1565, 1513, 1485, 1402, 1384, 1353, 1302, 1247, 1177, 1134,
1034, 800, 762 cm-1.
Inhibition of AChE
The inhibitory potency against AChE was evaluated by means of Ellman’s test [13]. AChE stock solution was
prepared by dissolving hAChE 0.5 unit in 100 mM PBS buffer (pH 7.4). The synthesized compounds 6a-j (10
μM) were dissolved in DMSO. The assay solution consisted of 100 mM PBS buffer (pH 7.4), with the addition
of 10 mM 5’5-dithio-bis-(2-nitrobenzoate) (DTNB, Sigma, D-8130), AChE (5 μL), drug (10 μL), and 12.5 mM
acetylthiocholine iodide (ATCh) water solution. The final assay volume was 900 μL. After incubation at 37ºC
for about 15 min, the mixture was added 50 mL ATCh and 50 mL DTNB, followed by incubation at 37ºC for 20
min with continuous gentle shake until the color change to yellow. Inhibitory effects were expressed as the
percentage of inhibition.
Docking studies
To disclose a possible binding mode of compounds 6a-j with hAChE enzyme’s binding pockets, the docking
simulation were performed using the available crystallographic structure of enzyme (PDB ID: 4EY7) [14] using
AutoDock 4.0. In order to obtain the better results from docking protocols, water molecules and other ligand
(NAG) were excluded. The polar hydrogen atoms of the enzyme were added, the non-polar hydrogen atoms
were merged and Gasteiger charges were assigned. For all ligands the non-polar hydrogen atoms were
merged, and the Gasteiger charges were assigned. The grid box dimensions were 60×60×60 Å around the
active site and the grid spacing was set to 0.375 Å. Docking was performed using the empirical free energy
function together with the Lamarckian genetic algorithm (LGA). The LGA protocol applied a population size
of 150 individuals, while 250,000 energy evaluations were used for the 20 LGA runs. In addition, the
maximum number of evaluations was set to 27,000. In this study, the co-crystal natural substrate, donepezil,
was taken out of the active site and docked again used as the template for molecular alignment.The cluster
analyses were computed with a cluster tolerance by less than 1.5 Å in positional root-mean-square
deviation. PyMol 1.5.0.3 software was used for visualization of protein-ligand interactions.
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Results and Discussion
Chemistry
The target compounds 6a-j were synthesized as illustrated in Scheme 1. At the first stage, anisaldehyde was
cyclized with N-acetylglycine in acetic anhydride, which led to a high yield of
4-(arylmethylene)-2-methyl-5(4H)-oxazolone 1. 1 converted to intermediate 2-Acetamido-3-
(4-methoxyphenyl)-2-propenoic acid 2 with acetone and water. Intermediate 2 was further hydrolyzed with
1 N hydrochloric acid to give 4-methoxyphenylpyruvic acid 3. The base-catalysed condensation reaction of
4-methoxyphenylpyruvic acid 3 with thiosemicarbazone in the presence of ethyl alcohol and 50 % NaOH,
followed by neutralisation in the presence of cold HCl that yielded
6-(4-methoxybenzyl)-3,4-dihydro-3-thioxo-1,2,4-triazin-5(2H)-one 4. Subsequently, the differently
substituted phenacyl chloride and 4 underwent intra-molecular cyclization on refluxing with glacial acetic
acid to yield 6-(4-methoxybenzyl)-3-hydroxylphenyl-7H-thiazolo[3,2-b] -1,2,4- triazin-7-ones 5a-b. There
after refluxing of the compounds (5a-b) with different substituted alkyl chloride in the presence of acetone
and K2CO3 provided 6-(4-methoxybenzyl)-3-aryl-7H-thiazolo [3,2-b]-1,2,4-triazin-7-ones 6a-j.
Inhibition of AChE
As shown in Table 1, 6-(4-methoxybenzyl)-3-aryl-7H-thiazolo [3,2-b]-1,2,4-triazin-7-one derivatives 6a-j
generally showed moderate to good hAChE inhibitory activities with inhibition values ranging from 37.80% to
77.82%. Among all the derivatives examined, compound 6i showed the most inhibitory activity and the
inhibition reached to 77.82%. However, all the synthesized compounds showed lower inhibitory activity than
the reference drug huperzine-A.
It was observed that the type and position of the substituents played an important role for the inhibitory
activities. As compared with the single substituent on the para-position of the phenyl group derivative 6a
and 6b (37.80% and 54.67%), the two substituents on the meta and para-position of the phenyl group
derivatives 6c and 6d showed increased activity (76.10% and 73.92%). On the other hand, the replacement
of the piperidinyl group of compound 6d and 6g by morpholinyl group (compound 6e and 6h) resulted in the
decreased activity.
Docking studies
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 model of the receptor was based on the crystal
structure of hAChE complex with donepezil (PDB ID: 4EY7). The co-crystal natural substrate was taken out of
the active site and docked again. The top 3 docking configurations were taken into consideration to validate
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the results and the RMSD was calculated for each configuration in comparison with the co-crystallized ligand,
donepezil. The acceptable results (RMSD within 0.61-0.83Å) indicated that the docked configurations have
similar binding positions and orientations within the binding site and are similar to the co-crystal structure,
which illustrates the fact that the docking protocols used could successfully generate the crystal
donepezil-AChE complex precisely. The pose (best conformation of docked donepezil)selection of docked
ligands was done based on the closest alignment to the original donepezil ligand as well as the interaction of
ligands with key amino acid residues such as Ser203, His447 at the CAS and Tyr72, Asp74, Trp286 at PAS. The
benzyl ring stacks against Trp86 near the active site, while the indanone ring stacks against Trp286 in the
peripheral anionic site.The docking simulations reveal all target compounds exhibited multiple binding
modes with the human AChE(Figure 2). The calculations with the flexible docking protocol placed all the
compounds correctly into the binding pocket as presented in Figure 3. The docking results revealed the three
compounds could suitably interact with the CAS and PAS of the enzyme. For example, the docking simulation
revealed that 4EY7 and compounds 6a, 6c and 6i interacted through π-π aromatic interactions and hydrogen
bonding (Figure 4). Both the phenyl ring of the compounds 6a, 6c and 6i may show π-π interactions with
Trp86. The nitrogen atom of side chain may also show cation-π interactions with Tyr341 (one of the six
residues of PAS clustered around the entrance to the active site gorge) with the distance of 4.6, 3.6 and 3.9
Å, respectively. Additional significant H-bond interactions were also observed between the oxygen atom of
6c with -OH group of Tyr133, Tyr124 and Tyr337 with the distance of 2.8 Å, 3.0 Å and 2.5 Å respectively.
Tyr133 and Tyr337 are important residues of “Anionic binding site” in the gorge which involved in optimally
positioning the ester at the acylation site along with binding to trimethylammonium choline through
cation-π interactions. For the compound 6i, more H-bond interactions with Tyr124, Trp86 and Tyr449 were
found, which might be the reason for its increased inhibitory potency.
Conclusion
Starting with anisaldehyde, a new series of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives were designed
and synthesized as dual binding site inhibitors of AChE. All these new derivatives have been investigated for
their inhibitory activity in vitro using Ellman’s method. The results revealed that most compounds with
moderate inhibitory activity. Among them, compounds 6i bearing diethylamino-2-oxoethoxy in the aryl ring
at C4 position exerts the most potency. Molecular docking results revealed that 6a, 6c and 6i might behave
as dual binding site inhibitors. Compound 6c with two substituents on the meta and para position of the
phenyl group show the better binding conformation than 6a. These studies suggest that
7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives with two substituents may be a promising structural
template for the development of novel AChE inhibitors in managing amnesic disorders including AD.
Conflict of Interest
The author(s) confirm that this article content has no conflicts of interest.
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Table 1. Inhibition of hAChE Activities by the targets at 10μM (n=2)
No.
6a
R1
H
R2
Inhibition (%)
37.80
6b
6c
H
54.67
76.10
CH3
6d
6e
CH3
CH3
73.92
56.26
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6f
CH3
CH3
CH3
66.14
6g
6h
70.48
43.06
6i
6j
CH3
CH3
77.82
71.82
huperzine-A
100.00
Figure 1. Structure of reported AChE inhibitors.
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Figure 2. Binding orientation of redocked co-crystal donepezil (blue) and original donepezil (red).
Figure 3. Superimposition of docking conformations of 6a (red), 6b (green), 6c (blue), 6d (yellow), 6e
(magenta), 6f (cyan), 6g (orange), 6h (white), 6i (salmon) and 6j (slate) inside the gorge of 4EY7 which were
showed as sticks. Only a part of residues of PAS (red), CAS (blue) and anionic binding site (yellow) of 4EY7
were showed as ball and stick to reveal the binding gorge. Some residues of protein were omitted for clarity.
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Figure 4. Docking of compounds 6a (A), 6c (B) and 6i (C) to 4EY7. The compounds 6a, 6c and 6i are colored
by green. The hydrogrn bonds, π–π and cation-π interactions are shown as red dashed lines.
Scheme 1. The synthetic route of 7H-thiazolo[3,2-b]-1,2,4-triazin-7-one derivatives
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