Design, synthesis, biological evaluation and molecular docking study of novel pyridoxine-triazoles as anti-Alzheimer's agents

Article abstract of DOI:10.1039/d0ra04942e

A series of multi-target natural product-pyridoxine based derivatives were designed, synthesized, characterized and evaluated as anti-Alzheimer agents. In vitro testing revealed the multi-functional properties of compounds such as inhibition of acetylcholinesterase (AChE), antioxidant and metal chelation. Among the series, 5i derivative was found most potent AChE inhibitor, possess antioxidant potential and chelating metal ions. Further binding interaction of 5i with AChE was studied using molecular docking, showed interaction with both PAS and CAS site of AChE. In silico predictions were also performed to predict toxicity and ADME properties of the molecule 5i and found within drug likeness range. Therefore, 5i could be a promising multi-functional compound that can be used for further development of novel drug for Alzheimer disease.

Full text of DOI:10.1039/d0ra04942e

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PAPER
Design, synthesis, biological evaluation and
molecular docking study of novel pyridoxine–
triazoles as anti-Alzheimer's agents†
Cite this: RSC Adv., 2020, 10, 26006
a
b
Tiyas Pal,a Saipriyanka Bhimaneni,b Abha Sharma
and S. J. S. Flora
*
*
A series of multi-target natural product-pyridoxine based derivatives were designed, synthesized,
characterized and evaluated as anti-Alzheimer agents. In vitro testing revealed the multi-functional
properties of compounds such as inhibition of acetylcholinesterase (AChE), antioxidant and metal
chelation. Among the series, 5i derivative was found most potent AChE inhibitor, possess antioxidant
potential and chelating metal ions. Further binding interaction of 5i with AChE was studied using
molecular docking, showed interaction with both PAS and CAS site of AChE. In silico predictions were
also performed to predict toxicity and ADME properties of the molecule 5i and found within drug
likeness range. Therefore, 5i could be a promising multi-functional compound that can be used for
further development of novel drug for Alzheimer disease.
Received 4th June 2020
Accepted 3rd July 2020
DOI: 10.1039/d0ra04942e
rsc.li/rsc-advances
showed the way to develop acetylcholinesterase (AChE) inhibitors
providing symptomatic relief to the AD patients. However, this
1 Introduction
age related disease poses a problem of uncontrollable bio-burden
of metal ions in the brain. This metal dyshomeostasis in turn
induces oxidative stress and formation of amyloid-b (Ab) plaques.
Due to this interconnection of etiologies, classical metal chelator
cannot do any benet to this existing problem of the disease.
Therefore MTDLs is needed in this juncture. Increase in bio-
metals amplies reactive oxygen species (ROS) which includes
augmentation of lipid peroxidation, protein oxidation, nitration
and glycol oxidation. Oxidative stress aggravates the pathophys-
iological conditions of neuronal damage.4 A molecule having
additional anti-oxidant potency along with AChE inhibitory
potential and metal chelating ability can be developed as
a potential anti-AD agent. Studies have stated that peripheral
anionic site (PAS) binding of AChE may promote Ab aggregation.
For PAS binding, aromatic rings with pi–pi stacking are recom-
mended as derived from the docking studies with donepezil.5
In light of the concepts mentioned above, and to focus on our
efforts to develop a new class of anti-Alzheimer's agents as
MTDLs, we focused on the synthesis of pyridoxine based 1,2,3-
triazoles and its biological evaluation. We assessed the synthe-
sized compounds for AChE inhibition, antioxidant potency,
metal chelation ability using in vitro assays as screening methods.
We predicted various pharmacokinetic properties and toxicity of
the best compound of the series using in silico tools.
Alzheimer's disease (AD) is the most challenging neurodegener-
ative disease affecting mostly the population above 65 years of
age. The perplexing nature of the disease makes it all more
difficult for its ministration. More than 50 million are suffering
globally and the number is likely to get hiked up to 150 million by
the year 2050.1 The multifaceted nature of the disease with
several interconnected etiologies led to the idea of developing
multi-target directed ligands (MTDLs). MTDLs in the eld of
neurodegenerative diseases is now a very signicant area of
research developed by academia and industry.2,3 The design of
MTDL comprises two or more pharmacophore moieties which
are responsible for targeting multiple targets which are
accountable for the pathogenesis of the disease. Therefore,
MTDLs are single molecules with multiple functions which are
strategically designed to serve the purpose of developing poten-
tial new chemical entities that can be used against AD.
The currently available Food and Drug Administration (FDA)
approved drugs mainly focus on the cholinergic hypothesis, as
one of the most excavated areas to slow down the progression of
AD. Cholinergic hypothesis, amyloid-b cascade hypothesis, metal
dyshomeostasis, tau hypothesis and oxidative stress hypothesis
are some of the identied etiologies responsible for this disease.
Damage to cholinergic neurons lead to cognitive decline which
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education
and Research, Raebareli, India. E-mail: abha.sharma@niperraebareli.edu.in
bDepartment of Regulatory Toxicology, National Institute of Pharmaceutical Education
and Research, Raebareli, India. E-mail: sjsora@hotmail.com
2 Results and discussion
2.1 Design
Nitrogen heterocycles have long been of medical interest6–8
while, triazoles falling under the category of nitrogen
† Electronic supplementary information (ESI) available: Spectra data. See DOI:
10.1039/d0ra04942e
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heterocycles have been the moiety of interest. Numerous
molecules with triazole moiety have been reported. In this
regard, Rastegari et al. reported 1,2,3-triazole chromenone car-
boxamide derivatives as AChE inhibitors.9 Jalili-Baleh et al.
developed 3-phenylcoumarin lipoic acid based triazole deriva-
tives as anti-Alzheimer's agents.10 Fig. 1 provides a summary of
few triazole and pyridoxine containing molecules reported
recently as potential anti-Alzheimer's agents.9–15
2.2 Chemistry
We synthesized pyridoxine based triazoles derivatives (Scheme
1). The target molecules were synthesized via four step reaction
scheme using pyridoxine hydrochloride (1) as starting material.
In the rst step, commercially available pyridoxine hydrochlo-
ride (1) was protected at the 3- and 4-hydroxy groups using p-
toluenesulfonic acid monohydrate and dry acetone in the
Pyridoxine is known to reduce homocysteine levels in Alz-
heimer's patients. Homocysteine is derived from amino acid
methionine and is a non-protein homologue of cysteine. The
directly attached hydroxyl group to the pyridine nucleus might
be playing a role in chelation of metal and also for the anti-
oxidant properties. Pyridoxine has successfully completed the
phase 3 clinical trials as a vitamin which can reduce homo-
cysteine levels in AD affected patients. The circulating high level
of homocysteine in AD is responsible for deposition of amyloid
b plaques.16,17 Pyridoxine–resveratrol have been reported as only
pyridoxine based compounds against AD. However, pyridoxine
based triazoles have not been reported and therefore, herein we
report this new class of compounds.
Taking into the consideration the multifaceted nature of the
disease, we designed pyridoxine based 1,2,3-triazoles as an
MTDL which might be an effective agents against AD. We have
designed our target molecules containing both the pyridoxine
and triazole moiety as a new class of MTDL using linking
approach. Fig. 2 provides the possible hypothesis behind the
designing of our target molecules.
Fig. 2 Design of target molecules chemistry.
Fig. 1 Triazole and pyridoxine containing molecules as anti-Alzheimer's agents.
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presence of 2,2-dimethoxypropane under argon atmosphere. inactive towards the enzyme. With the substitution of methyl
Then compound 2 was treated with propargyl bromide using group at the para position (5b) it however, showed inhibitory
NaH as base and dry THF as solvent under argon atmosphere to activity as compared to 5a. By changing the position of methyl
give compound 3. In the next step, various substituted benzyl group from para to meta (5c) and ortho (5d) position, a marked
azides were generated in situ from benzyl bromides followed by change was noted. A meta substitution of the electron donating
synthesis of triazole moiety aer addition of compound 3 using group (EDG) offered a better inhibitory activity almost twice
the classical CuAAC, generating compounds 4(a–q). Finally, potent as compared to 5b, while the ortho alkyl substitution
target compounds 5(a–q) were obtained through deprotection proved even better. Substitution of methoxy group at the 3rd
of the isopropylidene group.
position (5e) and at 3rd and 5th position of the aromatic ring (5f)
had no inhibitory activity, however, methyl substitution proved
better than these. A double methoxy substitution was found
more efficacious compared with its single substitution.
2.3 In vitro assay of AChE inhibition
We further explored by trying different halides as substitu-
ents and their effect on the inhibition on the enzyme. We also
synthesized F, Cl and Br derivatives at different position.
Halogens are supposed to have +R effect on the aromatic ring,
despite of their position. When the effect of halogen is observed
at the para position, uoro substituent (5g) showed lesser effi-
cacy compared to –Cl (5j) and –Br (5m) derivatives. However, we
did not nd a uniform trend in activity as we moved down the
periodic table. Chloro substitution at the para position was
more potent in comparison to bromo substitution. However,
meta substitution of halogens showed much better results as
compared to the para substitution. Change in position from
para to meta did not prove successful in improving potency in
chloro substitution (5k), as there was no such change in the IC50
value. Bromine at the 3rd position of the aromatic ring (5n) was
more active in comparison to the uoro substitution (5h). We
even tried to nd out the effect of substitution when two posi-
tions on the aromatic ring are occupied with the halogen atoms.
We thus included uorine in 3rd and 5th position (5i) and in
The in vitro activity of the new synthesized molecules was done
using AChE from electric eel (EeAChE) following Ellman's
method.25 All the molecules were tested for their ability to
inhibit AChE activity, keeping donepezil as the reference
compound. The IC50 values of the molecules are summarized in
Table 1. AChE inhibition remains a very critical end point in
slowing down the progression of the disease, and thus we
selected to determine the ability of our compounds to inhibit
the aforesaid enzyme.
2.3.1 Structure activity relationship. Our target molecule
contains triazole and pyridoxine moieties incorporated into it.
Before starting our investigation on our library of compounds
5(a–q), we sought to nd out the inhibitory activity of pyridoxine
on AChE and observed it to be inactive against the enzyme;
these results correlated well with the studies of Drtinova et al.18
We also sought to nd out the effect of triazole moiety and
substitution on the aromatic ring on enzymatic activity.
The unsubstituted 5a did not show much inhibition even at
the highest concentration (10 mM), thus can be considered
Scheme 1 Reagents and conditions-(i) 2,2-dimethoxypropane, p-toluenesulfonic acid monohydrate, anhydrous acetone, under argon atmo-
sphere 20 h, rt (ii) NaH, propargyl bromide, dry THF, reflux under argon atmosphere, 4 h (iii) benzyl bromide, NaN3, triethylamine, CuSO4$5H2O
(10 mol%), ascorbic acid (25 mol%), t-BuOH : H2O (1 : 1), reflux for 6–12 h (iv) THF, 10% conc. sulphuric acid, 70 ꢀC, 6–12 h.
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Table 1 Results of AChE inhibition and anti-oxidant values
Compound
Ar
EeAChE IC50 ꢁ SEMa (mM)
ORAC index ꢁ SEMb
5a
>10
1.0564
1.8428
ꢁ0.3604
ꢁ0.0980
5b
5c
3.3833
ꢁ0.0927
1.3759
ꢁ0.1019
ꢁ0.0924
2.0164
1.9723
ꢁ0.0538
ꢁ0.4236
5d
5e
0.9073
4.5737
ꢁ0.0995
ꢁ0.1694
1.8862
2.3425
ꢁ0.2533
ꢁ0.0228
5f
2.6508
5g
5h
6.4687
ꢁ0.1334
ꢁ0.0227
1.4489
ꢁ0.1579
ꢁ0.0315
1.5437
2.1015
5i
5j
1.5609
ꢁ0.0237
ꢁ0.0435
1.2138
ꢁ0.2832
ꢁ0.1800
2.7187
1.9697
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Table 1 (Contd.)
Compound
Ar
EeAChE IC50 ꢁ SEMa (mM)
ORAC index ꢁ SEMb
5k
2.70723333
ꢁ0.0127
0.8358
ꢁ0.0374
5l
3.3777
3.1383
1.0801
6.5162
3.6525
ꢁ0.2162
ꢁ0.0432
ꢁ0.0851
ꢁ0.1326
ꢁ0.0120
1.9421
1.215
ꢁ0.1603
ꢁ0.1383
ꢁ0.1003
ꢁ0.1775
ꢁ0.2761
5m
5n
5o
5p
2.2079
2.0758
0.8718
5q
1.1346
ꢁ0.1632
0.7168
ꢁ0.1410
ꢁ0.1084
Pyridoxine
Donepezil
Trolox
—
—
—
n.ac
0.5249
N.Td
1
<5 mM
N.Td
a
IC50 values is the concentration of the enzyme required to decrease the enzyme activity by 50% and are the mean of three independent
b
experiments, represented in mean ꢁ SEM (SEM ¼ standard error mean). The mean ꢁ SEM of three independent experiments. Data are
represented as ORAC-FL values of Trolox equivalents (mM of tested compound/mM of Trolox). c n.a ¼ not active. d N.T ¼ not tested.
another compound, chlorine at 3rd and 4th position (5l). Inclu- inhibition between the two can be due to the high electroneg-
sion of two uorine atoms at both the meta positions, did not ativity and small size of uorine.
show change in potency but there was a spurt in % of enzyme
We tried with triuoro-methyl substitution at para, meta and
inhibition. To summarize the effect of di-substitution on the ortho positions (5o, 5p, 5q). Triuoro-methyl substitution at the
aromatic ring, 5i was considered the best as compared to 5l and ortho position was the best amongst the three both from the
5f. Owing to the fact that uorine and methoxy groups are both perspective of potency and inhibitory activity. However, when
ring activators, the difference in potency and percentage of compared with methyl substitution, 5b was double potent in
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Table 2 Percentage of AChE inhibition of 5c, 5d, 5h, 5i, 5n, 5q and donepezil at different concentrations of the compoundsa
156 mM
312 mM
625 mM
1.25 mM
%
2.5 mM
%
5 mM
%
10 mM
%
%
ꢁSEM
%
ꢁSEM
%
ꢁSEM
ꢁSEM
ꢁSEM
ꢁSEM
ꢁSEM
5c
5d
5h
5i
5n
5q
Donepezil
22.49
33.53
19.04
14.97
14.69
22.49
89.07
3.62
6.44
0.29
4.12
1.15
3.62
1.19
26.92
36.41
25.49
26.58
25.79
26.92
94.5
0.55
6.8
39.11
51.15
35.1
35.38
41.99
39.11
97.44
2.48
5.55
1.59
0.47
1.42
2.48
0.73
53.8
3.13
4
64.11
69.57
60.48
61.41
70.16
64.11
99.7
1.79
2.3
73.43
81.3
1.13
2.36
0.26
0.1
2.44
1.13
0.69
81.92
86.03
69.02
77.89
86.28
81.92
99.95
0.85
0.63
0.36
0.15
1.38
0.85
0.48
58.56
56.37
43
57.39
53.8
2.22
3.92
2.73
0.55
0.81
1.85
1.52
4.99
3.13
0.93
1.24
2.82
4.11
1.79
1.04
66.56
66.78
75.88
73.43
99.74
100.88
a
Data presented here is of % of AChE inhibition mean ꢁ SEM of three independent experiments at different concentrations. Donepezil is taken as
the reference compound.
respect to 5o. We got the same observation for meta substitution
as well, 5c was twice in terms of potency as compared to 5p.
Methyl substitution at the ortho position was marginally better
in terms of triuoro-methyl substitution at the same position.
Methyl group is considered as ring activator, while triuoro-
methyl group as ring deactivator. Therefore, the observations
conrm that EDG is best suited for inhibitory activity against
AChE, when pyridoxine based triazoles are concerned. Table 2
gives a detailed information of percentage of inhibition of AChE
at different concentrations of by 5c, 5d, 5h, 5i, 5n, 5q and the
reference compound, donepezil. Fig. 3 shows the comparative
results of percentage of inhibition of AChE by 5c, 5d, 5h, 5i, 5n,
5q derivatives at 10 mM concentration, taking donepezil as the
reference standard.
To summarize all our observations with respect to our stan-
dard compound donepezil, our molecules required a higher
concentration to show their inhibitory activity. Donepezil showed
almost 89% inhibition at the highest dilution in which we tested
our compounds (156 mM) and 65% inhibition at 5 mM. However,
at the highest concentration, that is, at 10 mM concentration,
compounds showed a maximum of 86% inhibitory activity
towards AChE while donepezil exhibited 100% activity.
2.4 Antioxidant study
Aging is associated with oxidative stress resulting in generation of
reactive oxygen species. We determined the oxygen radical
absorption capacity-uoroscein (ORAC-FL) values for the entire
series. The ORAC-FL values are given in Table 1 and are
a measure of the total antioxidant capacity. We generated peroxyl
free radicals using the free radical generator 2,2-azobis(2-
amidino-propane)dihydrochloride (AAPH) and uorescein as
the uorescent probe in this assay at 37 ꢀC. The total antioxidant
capacity of the molecules were determined in equivalents to
Trolox (vitamin E analogue), keeping both at same concentration
(8 mM). One of the reasons for choosing pyridoxine as a phar-
macophore was for it's antioxidant nature. We observed all our
compounds to have anti-oxidant property as shown in Table 1.
Vitamin B6 being a dietary supplement was tested using the
ORAC-FL method. All the compounds showed almost similar
anti-oxidant nature, however, pyridoxine showed the most effi-
cacy in terms of an anti-oxidant. Pyridoxine was taken as a refer-
ence compound to evaluate the entire series in this experiment.
Therefore, from the observations of Table 1, we selected 5c,
5d, 5h, 5i, 5n and 5q on the basis of AChE inhibitory activity and
antioxidant property of the compounds for further evaluations.
2.5 Metal chelation study
Chelation of bio-metals will make the compounds prove as
MTDLs and be an added advantage. We tested 5c, 5d, 5h, 5i, 5n
and 5q against bio-metals Fe3+, Cu2+, Zn2+, Al3+ which are
responsible for AD, using UV spectrophotometer within the
wavelength range of 200 to 600 nm. All these bio-metals are
responsible for the pathophysiological conditions in AD. We
found a marked ability within the compounds to chelate Fe3+
.
Compounds 5c, 5i and 5q chelated both Fe3+ and Al3+, 5i
chelated the metals most with a 222% increase in absorbance at
292 nm which also includes a bathochromic shi of about 3 nm
in case of Fe3+ and an increase of 104% in absorbance at 292 nm
which too included a bathochromic shi of 4 nm in case of Al3+
.
The observations for all the compounds studied for metal
chelation a study has been tabulated in Table 3.
Although, aluminium being the most abundant metal on
the earth's crust, it do not pose an immediate health concern
when being exposed to the human body. Chronic exposure to
Fig. 3 Percentage of inhibition of AChE by 5c, 5d, 5h, 5i, 5n, 5q
derivatives at 10 mM concentration. Data presented here is mean ꢁ
SEM of three independent experiments.
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aluminium in drinking water has been studied for it's asso- form an essential part of our system. We observed the charac-
ciation with AD. To add more to the pathophysiology, teristic curve of 5i–Fe3+ as 5i showed affinity towards Fe3+ in the
aluminium is known to enhance ROS generation and stimu- metal pool (Fig. 4b).
lated iron based generation of ROS species. Both these metals
We studied the stoichiometric association of the Fe3+–5i
are also known to be associated with the senile plaques.19 complex by keeping the amount of ligand xed and titrating the
Therefore, 5i has the ability to chelate both the trivalent amount of metal in increasing concentration ranging from 0–
cations which can add well to the property of the compound as 100 mM using UV spectrophotometer. The spectral changes were
a MTDL as shown in Fig. 4a.
recorded at nm and a graph was plotted (Fig. 4c). There was an
The main challenge for chelation therapy of metals is increase in intensity at 293 nm and a decrease at 327 nm, on
selectivity towards metal cations. So we sought to check the successive increase in concentration of Fe3+. We analysed the
selectivity towards essential metals responsible for proper data and plotted the Job's plot, at 239 nm and 327 nm where on
physiological functions of our body. We included Ca2+, Mg2+, extrapolation we got a mole fraction of 0.5 in both the wave-
Na+ and K+ in our study and interestingly found out that 5i did length conrming formation of 1 : 1 stoichiometric complex
not chelate these metal ions at all (Fig. 4a). The spectrum of 5i– between 5i and Fe3+ (Fig. 4d and e).
Fe3+ complex was quite different from the parent spectrum and
Good et al. performed a laser microprobe study or the
those by other metals. Owing to the fact, that our biological LAMMA study where it was found selective deposition of Al
system comprise a mixture of metal ions, we sought to check the and Fe in the neurobrillary tangles.20 Therefore, 5i being
affinity of our compound 5i towards Fe3+ in the presence of a selective aluminium and iron chelator can be a potential
other metals (Fe3+, Zn2+, Cu2+, Ca2+, Mg2+, Na+ and K+) present inhibitor against these potentially toxic metals and can
in equal amount. However, we excluded Al3+ from the study as surely halt the mechanism of pathological important
the purpose was to check affinity towards Fe3+ and Al3+ does not structures.
Table 3 Observations from metal chelation studies
Cu2+
Zn2+
Al3+
Fe3+
Compound
% DAa
Shib
% DAa
Shib
% DAa
Shib
% DAa
Shib
5c
lmax
256.39
287.7
326.31
0
0
0
0
0
0
0
0
0
0
0
0
—
(+)58.41
(+)141.5
—
—
—
(ꢂ)4.76
(ꢂ)171.14
(ꢂ)6.22
(+)0.42
—
—
5d
lmax
254.28
288
324.35
(+)8.76
(ꢂ)34.63
(+)3.75
(ꢂ)1.21
0
(+)0.79
(+)6.04
(ꢂ)1.21
(ꢂ)1
0
(+)9.36
(ꢂ)1.21
(ꢂ)2
(+)48.04
(ꢂ)1.19
(ꢂ)21.00
0
(ꢂ)34.62
(ꢂ)31.94
(ꢂ)4
(ꢂ)8.41
(ꢂ)7.13
(ꢂ)0.41
0
5h
lmax
257.15
288.5
325.74
(ꢂ)10.20
(ꢂ)9.15
(+)5.65
(+)1.03
(ꢂ)0.5
(+)0.74
(+)17.14
(+)20.42
(+)11.01
(+)0.31
(ꢂ)0.36
(ꢂ)0.26
(+)11.02
(+)3.52
(+)10.42
0
(ꢂ)65.31
(ꢂ)105.63
(ꢂ)27.08
(+)0.67
(+)0.67
(+)0.36
(+)1.4
(ꢂ)0.26
5i
lmax
257.85
288.78
325.93
0
0
0
0
0
0
0
0
0
0
0
0
—
—
—
—
(ꢂ)104.57
(ꢂ)3.68
(ꢂ)221.83
(ꢂ)2.6
(+)43.01
(ꢂ)1.09
(+)48.38
(ꢂ)3.25
5n
lmax
254.72
290.57
324.71
(+)17.34
(+)18.93
(+)16.59
(ꢂ)1.09
(+)0.67
0
(+)1.24
(+)2.43
(+)4.255
(ꢂ)1.08
(+)2.07
(ꢂ)0.67
(+)5.88
(+)5.82
(+)4.25
(ꢂ)1.08
(+)2.07
(ꢂ)0.67
(ꢂ)54.80
(ꢂ)70.39
(ꢂ)23.62
(ꢂ)0.72
(+)2.07
(+)0.36
5q
lmax
258.23
288.46
325.56
0
0
0
0
0
0
0
0
0
0
0
0
—
—
—
—
(ꢂ)78.07
(ꢂ)3.62
(ꢂ)185.50
(ꢂ)2.92
(+)39.70
0
(+)47.94
0
a
Data represented here is percentage change in absorbance; (+) indicating a decrease in absorbance and (ꢂ) as increase; calculated by the formula
[(abs of tested compound ꢂ abs of complex)/abs of tested compound] ꢃ 100. b Data represented here is shi in nm; (+) indicates a hypsochromic
shi and (ꢂ) indicates a bathochromic shi as calculated from the formula [lmax of tested compound ꢂ lmax of complex].
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Fig. 4 (a) Metal chelation spectra of 5i. (b) Selectivity spectrum of 5i from metal pool comprising of Fe3+, Zn2+, Cu2+, Ca2+, Mg2+, Na+ and K+; 5i
formed complex with Fe3+ as identified from it's characteristic curve shown in (a). (c) Titration spectra of Fe3+ with 5i with 20 mL aliquots of metal
solution to in increase the concentration of Fe3+ from 0–100 mM [a–k], with successive increment of 10 mM. (d) Job's plot of 5i at 293 nm. (e)
Job's plot of 5i at 327 nm.
the other hand, pyridine nucleus of the pyridoxine moiety was
seen to have p–p interaction with hydrophobic Trp 286. The
principal amino acid of PAS, that is, Trp 86, was found in the
vicinity of the ligand.22 The hydroxyl group of the pyridoxine was
found to form hydrogen bond with Arg 296. The catalytic
anionic site (CAS) amino acids Ser 203 and His 447 were found
in close proximity of the ligand. The dock score of 5i calculated
2.6 In silico study
2.6.1 Molecular docking study. We investigated on the
binding mode of pyridoxine based triazole derivatives with
AChE. We studied the binding interaction of 5i with AChE using
molecular docking in Schrodinger glide. The Fig. 5 represents
the molecular interactions of 5i with the active site amino acid
residues of the enzyme AChE. The molecular docking was done
with recombinant human AChE (PDB ID 4EY5) and it's 2D
representation has been shown in Fig. 5.21 The interactions
mainly observed were with the peripheral anionic site of the
enzyme (PAS site) with hydrophobic amino acids Tyr 124 and
Phe 338 having p–p interactions with triazole nucleus of 5i. On
from the molecular docking study was ꢂ8.50 kcal Jꢂ1
.
The ligand showed interactions with the PAS site amino
acids of AChE and binding to the PAS site of the enzyme is
known to inhibit Ab aggregation due to the homologous
domains of the b-neurexin with PAS site, itself serving as
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Fig. 5 2D representation of molecular docking study of 5i with AChE (PDB ID 4EY5).
a ligand. Thus in silico results of molecular docking with ligand
validated the results obtained from the in vitro AChE properties of the molecule 5i. The gure displays two parts, the
experiments. white part representing those set of values which allows gastric
Fig. 6b is the BOILED EGG representation of the permeation
2.6.2 Predictive ADME properties of 5i by computational permeation and the yolk or the yellow region representing those
methods. Taking the help of SwissADME (http:// set of values which allows blood–brain-barrier permeation. The
www.swissadme.ch), we predicted the drug-like properties, graph is plotted against W log P[a log P(n-octanol/water parti-
pharmacokinetic and physicochemical properties of 5i. This tion coefficient) method developed by Wildman and Crippen]
open access web tool comprised procient methods of predic- versus TPSA or Topological Polar Surface Area in the BOILED
tive tools like BOILED EGG,23 Bioavailability Radar. The so- EGG representation. The blue dot in the gure written as
ware presents an early estimation of these ADME parameters Molecule 1 is of 5i.
(for Absorption, Distribution, Metabolism and Excretion) and
2.6.3 Toxicity prediction of 5i by computational methods.
provides sufficient light to the physicochemical parameters The in silico toxicity prediction can reduce the time, cost and the
which is needed to be improved during formulation. The in number of animal experiments. We used the ProTox soware
silico result predicted by the aforesaid soware is presented in for this in silico study, which identies the LD50 value of the
Fig. 6a and b. The molecule 5i was found to possess good drug median lethal dose of a drug aer analysing the similarity of the
likeness properties and was well within the Bioavailability compounds and also identies the toxic fragments.25 It is an
Radar. The Bioavailability Radar gives a rst glance of the drug- open access web tool (http://www.tox.charite.de/tox), which
likeness of the molecule. The spider web like structure given displays strong results at (sensitivity, specicity and precision of
inside Fig. 6a is the Bioavailability Radar. The pink region is the 76, 95 and 75%, respectively) which are validated by diverse
optimal range for every property. Daina et al. explained well external set. Fig. 7 displays the oral toxicity prediction results
about the web tool for prediction of drug likeness properties. All which show the molecule 5i to have a predicted LD50 of 3350 mg
the predicted properties of the compound 5i by the web tool kgꢂ1, falling under the Class 5 of toxicity of chemical classi-
have been put up in Fig. 6a.24
cation. The molecule is predicted to be inactive in case of
hepatotoxicity, carcinogenicity, mutagenicity, cytotoxicity, all
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Fig. 6 (a) Predicted ADME properties of 5i as obtained from the report generated by SwissADME software. (b) BOILED EGG representation of 5i
(denoted as Molecule 1 and represented by blue dot in the figure). From the BOILED EGG representation, the yolk denotes the points which
allows passive blood–brain-barrier permeation, while the white region denotes points for passive gastrointestinal tract absorption. The graph is
plotted against W log P[a log P(n-octanol/water partition coefficient) method developed by Wildman and Crippen] versus TPSA or Topological
Polar Surface Area.
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Fig. 7 Report of toxicity prediction as generated by ProTox-II online tool.
nuclear signalling pathways and stress response pathways. The a challenge in metal chelation therapeutics, henceforth, further
predicted LD50 value of 5i is well in correlation with the fact that throwing light on the ability of 5i to selectively chelate Fe3+ from
pyridoxine itself rarely causes toxicity when given in very high a pool of metal ions, gives it an added advantage. None of the
doses. The in silico results generated gives us intuitive idea that literature of triazole based anti-Alzheimer's agents have re-
5i can be considered as a much safer molecule.
ported selectivity towards metal ions with a drastic change in
absorbance. We concluded our work with in silico studies of
drug likeness, pharmacokinetic properties of 5i, along with
toxicity predictions which predicted it to be under Class 5
according to the toxicity labelling of chemicals. Our initial
ndings can open up new therapeutic areas in multi-target
directed ligands against AD.
3 Conclusion
Here, we report the design, synthesis and biological evaluations
of a novel class of pyridoxine based triazole as multi-target
directed ligands. Pyridoxine itself is very crucial for the homo-
cysteine balance in AD patients and as well as considered an
important vitamin for our daily diet. However, as proved from
our experiment, it is not an AChE inhibitor. Out of the seven-
teen synthesized pyridoxine based triazoles, six showed good
AChE inhibition and antioxidant potency. Meta and ortho
4 Experimental section
4.1 Chemistry
4.1.1 General remarks. All chemical reagents and solvents
substitution on the aromatic ring with EDG or ring activator were purchased from Sigma Aldrich, Spectrochem Pvt. Ltd, Alfa
proved beneciary for AChE activity. With further metal chela- Aesar and used without further purication. Reactions were
tion studies, we found 5i as the best compound of this new monitored by Thin Layer Chromatography (TLC) using Merck
series. 5i has good AChE inhibitory activity (IC50 ¼ 1.56 ꢁ 0.02 silica gel 60 F254 pre-coated plates (0.25 mm) and the
mM) with 77.89% of inhibition at highest concentration and compounds were examined under UV chamber (Ultra violet
possessed antioxidant property having ORAC-FL value of 1.21 ꢁ uorescence analysis cabinet-PSI). Column chromatography
0.28 equivalent to Trolox, whose ORAC-FL value is taken as 1. was performed with silica gel (100–200 mesh). All products were
The in vitro results for 5i has been validated with the help of characterized by 1H NMR, 13C NMR and IR spectroscopy. 1H, 19
F
molecular docking with AChE by investigating it's binding and 13C NMR were recorded on JEOL RESONANCE ECZ500R at
poses. The compound 5i chelated Fe3+ as the spectra showed 500 MHz, 470 MHz and 125 MHz respectively with chemical
about 220% increase in absorbance and a 104% increase was shi (d) expressed in parts per million in CDCl3 at 27 ꢀC
seen in case of Al3+, exhibiting bathochromic shi in both. downeld from TMS as an internal standard and coupling in
Selectivity towards targeted metal ion has always been Hertz (Hz). Splitting patterns are described as singlet (s),
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doublet (d), triplet (t), multiplet (m), doublet of doublet (dd) and prepared via the reaction of benzyl bromides (1.1 mmol) with
doublet of triplet (dt). For 1H NMR, 13C NMR reference for sodium azide (1 mmol) in the presence of triethylamine in t-
CDCl3 appeared at 7.26 and 77.367 ppm respectively. For 1H BuOH/water (1 : 1). Aer around 30 min, 2,2,8-trimethyl-5-
NMR, 13C NMR reference for DMSO-d6 appeared at 2.50 ppm ((prop-2-yn-1-yloxy)methyl)-4H-[1,3]dioxino[4,5-c]pyridine (3) (1
and 39.52 ppm respectively. IR spectra were recorded on mmol) in absolute t-BuOH (1 mL) was added to the solution in
a BRUKER ECO-ATR Spectrometer. HRMS was measured on LC- the presence of CuSO4$5H2O (10 mol%ꢀ) and freshly prepared
ꢀ
MS by Agilent Technologies LC-MS and purity check was done ascorbic acid solution (25 mol%), at 50 C and stirred at 70 C
using HPLC on a HPLC by Agilent Technologies. The retention for 12 h. Aer completion of the reaction (checked by using
time in HPLC is represented as tR and calculated in minutes TLC), the reaction mixture was passed through a slurry of Celite
(min).
in ethyl acetate. The ltrate was concentrated under reduced
4.1.2 General procedure for the acetonide protection of (5- pressure. The organic residue was extracted with EtOAc (20 mL
hydroxy-6-methylpyridine-3,4-diyl)dimethanol. To a suspension ꢃ 3). The organic phase was washed with brine and dried over
of pyridoxine hydrochloride (5.00 g, 24.3 mmol) in dry acetone anhydrous Na2SO4. The solvent was evaporated to dryness
(75 mL) under argon atmosphere, added 2,2-dimethoxypropane under reduced pressure to afford compounds 4(a–q). The
(50.1 mL, 408 mmol) and stirred for 10 min.26 Following this, p- compounds were nally washed with hexane.
toluenesulfonic acid monohydrate (18.5 g, 97.2 mmol) was
4.1.5 General procedure for deprotection of compounds
added and the reaction was stirred in room temperature for 20 4(a–q). The corresponding compounds 4(a–q) (0.60–0.65 mmol)
hours. Completion of the reaction was monitored by TLC. The was dissolved in THF (5 mL), and 10% HCl solution (4 mL) was
reaction mixture was neutralized with aqueous sodium bicar- added. The reaction mixture was reuxed for 6–11 h under an
bonate, concentrated under reduced pressure, and the dark argon atmosphere. Aer complete reaction, THF was evapo-
brown solution was extracted with dichloromethane (40 mL ꢃ rated under reduced pressure. The residue was neutralized with
3). The organic phase was washed with brine and dried over aqueous saturated sodium bicarbonate solution, and extracted
anhydrous Na2SO4. The DCM layer was concentrated in vacuum with EtOAc (20 mL ꢃ 3). The organic phase was washed with
to provide a crude product, which was washed with hexane to brine and dried over anhydrous Na2SO4. The solvent was
give compound 2 as white crystals in 86% yield.
evaporated to dryness under reduced pressure to 5(a–q) in 85–
96% yield. The compounds were nally washed with hexane.
4.1.5.1 5-(((1-Benzyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)-4-
4.1.2.1 (2,2,8-Trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)
methanol (2). White solid crystals; mp-110–113 ꢀC; Rf ¼ 0.5 (ethyl
1
acetate/hexane 3 : 2); H NMR (500 MHz, chloroform-d) d 7.77 (hydroxymethyl)-2-methylpyridin-3-ol (5a). Brown oil; yield 95%;
(s, 1H, N]CH) d 4.91 (s, 2H, O–CH2) d 4.52 (s, 2H, CH2–OH) Rf ¼ 0.4 (DCM/methanol 19 : 1); 1H NMR (500 MHz, chloroform-
d 2.34 (s, 3H, CH3) d 1.53 (s, 6H, 2 ꢃ CH3); 13C NMR (125 MHz, d) d 7.75 (s, 1H, N]CH) d 7.39 (s, 1H, C]CH–N) d 7.36–7.31 (m,
chloroform-d) d 145.44, 145.09, 138.42, 130.59, 125.24, 99.41, 3H, Ar–H) d 7.21 (dd, J ¼ 7.2, 2.2, 2H, Ar–H) d 5.45 (s, 2H, N–CH2)
58.24, 57.88, 24.53, 18.24; IR (cmꢂ1) n: 3851.17, 3619.70, d 4.95 (s, 2H, CH2–O) d 4.43 (s, 2H, CH2–OH) d 4.43 (s, 2H, CH2–
1695.98, 1213.27.
O) d 2.40 (s, 3H, N–C–CH3); 13C NMR (125 MHz, chloroform-d)
4.1.3 General procedure for synthesis of 2,2,8-trimethyl-5- d 152.05, 148.52, 144.80, 139.90, 134.26, 131.57, 129.28, 128.21,
((prop-2-yn-1-yloxy)methyl)-4H-[1,3]dioxino[4,5-c]pyridine. To 127.54, 122.73, 68.25, 62.28, 59.77, 54.38, 18.67; IR (cmꢂ1) n:
a suspension of sodium hydride (1.5 eq.) in THF (40 mL), added 3141.08, 2926.77, 2863.95, 2363.77, 2246.90, 1615.93, 1225.02,
(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)methanol (2) 1213.27; HRMS (m/z): calculated for C18H20N4O3 [M + H]+
(1.00 g, 4.7 mmol) under argon atmosphere and reuxed for 341.1691; found, 341.1611; HPLC purity-99%; tR ¼ 9.86 min.
30 min. Following this, propargyl bromide (2 eq.) was added
4.1.5.2 4-(Hydroxymethyl)-2-methyl-5-(((1-(4-methylbenzyl)-
dropwise for 1 h and the reaction mixture was reuxed for 4 h. 1H-1,2,3-triazol-4-yl)methoxy)methyl)pyridin-3-ol (5b). Yellow
Completion of the reaction was monitored by TLC. The reaction sticky solid; yield 88%; Rf ¼ 0.4 (DCM/methanol 19 : 1); 1H NMR
mixture was concentrated under reduced pressure, quenched (500 MHz, chloroform-d) d 7.83 (s, 1H, N]CH) d 7.35 (s, 1H, C]
with saturated ammonium chloride solution and extracted with CH–N) d ¼ 7.14 (q, J ¼ 8.1, 4H, Ar–H) d 5.42 (s, 2H, N–CH2) d 4.97
dichloromethane (40 mL ꢃ 3). The organic phase was washed (s, 2H, CH2–OH) d 4.52 (s, 2H, CH2–O) d 4.42 (s, 2H, CH2–O)
with brine and dried over anhydrous Na2SO4. The DCM layer d 2.45 (s, 3HN–C–CH3), d 2.33 (s, 3H, Ar–CH3); 13C NMR (125
was concentrated in vacuum to provide a crude product, which MHz, chloroform-d) d 144.76, 140.56, 139.15, 134.72, 131.53,
was puried by column on silica using 20 : 80 ratio of hexane– 131.29, 130.05, 129.94, 128.28, 122.42, 68.35, 61.47, 59.67,
DCM solvent system to provide compound 3 in 75% yield.
54.22, 21.23, 18.85. IR (cmꢂ1) n: 3145.19, 2930.77, 2869.75,
2367.37, 2248.92, 1620.39, 1221.03, 1210.28; HRMS (m/z):
4.1.3.1 2,2,8-Trimethyl-5-((prop-2-yn-1-yloxy)methyl)-4H-[1,3]
dioxino[4,5-c]pyridine (3). Brown oil; Rf ¼ 0.6 (DCM/methanol calculated for C19H23N4O3 [M + H]+ 355.1770; found, 355.1772;
12 : 1); 1H NMR (500 MHz, chloroform-d) d 7.99 (s, 1H, N] HPLC purity-97%; tR ¼ 11.20 min.
CH) d 4.9 (s, 2H, O–CH2) d 4.71 (s, 2H, CH2–OH) d 4.46 (s, 2H,
4.1.5.3 4-(Hydroxymethyl)-2-methyl-5-(((1-(3-methylbenzyl)-
CH2–C) d 2.4 (s, 3H, CH3) d 1.5 (s, 6H, 2 ꢃ CH3); IR (cmꢂ1) n: 1H-1,2,3-triazol-4-yl)methoxy)methyl)pyridin-3-ol (5c). Yellow oil;
3292.03, 2992.75, 2931.93, 2858.50, 2114.38, 1067.29, 1025.50.
yield 87%; Rf ¼ 0.4 (DCM/methanol 19 : 1); 1H NMR (500 MHz,
4.1.4 General procedure for synthesis of substituted 5-(((1- chloroform-d) d 7.83 (s, 1H, N]CH) d 7.37 (s, 1H, C]CH–N)
benzyl-1H-1,2,3-triazol-4-yl)methoxy)methyl)-2,2,8-trimethyl-
4H-[1,3]dioxino[4,5-c]pyridine 4(a–q). Different derivatives were d 7.05–7.02 (m, 2H, Ar–H) d 5.42 (s, 2H, N–CH2) d 4.97 (s, 2H,
d 7.24–7.21 (m, 1H, Ar–H) d ¼ 7.16 (d, J ¼ 7.6 Hz, 1H, Ar–H)
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CH2–OH) d 4.52 (s, 2H, CH2–O) d 4.43 (s, 2H, CH2–O) d 2.45 (s, MHz, chloroform-d) d 148.67, 145.34, 140.30, 131.58, 130.16,
3H, N–C–CH3), d 2.32 (s, 3H, Ar–CH3); 13C NMR (125 MHz, 130.09, 122.49, 116.41, 116.24, 68.38, 61.78, 59.64, 53.64, 18.75;
chloroform-d) d 140.38, 139.18, 134.11, 131.64, 129.78, 129.16, 19F NMR (470 MHz, chloroform-d) d ꢂ112.10 to ꢂ112.17 (m); IR
128.97, 127.45, 125.67, 125.31, 122.58, 68.34, 61.68, 59.60, (cmꢂ1) n: 3247.63, 2896.70, 2912.86, 2378.87, 2367.85, 1613.10,
54.43, 21.39, 18.75. IR (cmꢂ1) n: 3146.20, 2931.47, 2868.75, 1245.81, 1218.23; HRMS (m/z): calculated for C18H20N4O3F [M +
2369.58, 2249.22, 1622.19, 1223.10, 1211.32; HRMS (m/z): H]+ 359.1519; found, 359.1514; HPLC purity-99.8%; tR
¼
calculated for C19H23N4O3 [M + H]+ 355.1770; found, 355.1768; 10.32 min.
HPLC purity-96%; tR ¼ 10.30 min.
4.1.5.8 5-(((1-(3-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
methyl)-4-(hydroxymethyl)-2-methylpyridin-3-ol (5h). Yellow oil;
4.1.5.4 4-(Hydroxymethyl)-2-methyl-5-(((1-(2-methylbenzyl)-
1H-1,2,3-triazol-4-yl)methoxy)methyl)pyridin-3-ol (5d). Dark- yield 86%; Rf ¼ 0.4 (DCM/methanol 19 : 1); 1H NMR (500 MHz,
green semisolid; yield 93%; Rf ¼ 0.4 (DCM/methanol 19 : 1); chloroform-d) d 7.81 (s, 1H, N]CH) d 7.42 (s, 1H, C]CH–N)
1H NMR (500 MHz, chloroform-d) d 7.83 (s, 1H, N]CH) d 7.29 d 7.33 (td, J ¼ 8.0, 5.9 Hz, 1H, Ar–H), d 7.06–6.99 (m, 2H, Ar–H)
(s, 1H, C]CH–N) d ¼ 7.30–7.25 (m, 1H, Ar–H), d 7.20 (dd, J ¼ d 6.92 (d, J ¼ 9.1 Hz, 1H, Ar–H) d 5.47 (s, 2H, N–CH2) d 4.98 (s,
7.2, 3.6 Hz, 2H, Ar–H), d 7.13 (d, J ¼ 8.0, 1H, Ar–H) d 5.48 (s, 2H, 2H, CH2–OH) d 4.51 (s, 2H, CH2–O) d 4.45 (s, 2H, CH2–O) d 2.44
N–CH2) d 4.97 (s, 2H, CH2–OH) d 4.52 (s, 2H, CH2–O) d 4.42 (s, (s, 3H, N–C–CH3); 13C NMR (125 MHz, chloroform-d) d 151.98,
2H, CH2–O) d 2.45 (s, 3H, N–C–CH3), d 2.24 (s, 3H, Ar–CH3); 13
C
148.68, 145.06, 140.25, 131.55, 131.00, 130.94, 127.45, 123.65,
NMR (125 MHz, chloroform-d) d 148.80, 143.86, 137.03, 132.11, 122.69, 116.15, 115.99, 115.24, 115.07, 68.37, 61.86, 59.69,
131.21, 129.61, 129.44, 126.83, 68.39, 61.62, 59.57, 52.56, 19.04; 53.72, 18.73; 19F NMR (470 MHz, chloroform-d) d ꢂ111.20 (ddd,
IR (cmꢂ1) n: 3147.20, 2930.70, 2870.59, 2372.85, 2247.31, J ¼ 9.0, 6.3, 2.2 Hz); IR (cmꢂ1) n: 3245.63, 2893.70, 2911.86,
1620.20, 1221.70, 1215.36; HRMS (m/z): calculated for 2375.77, 2377.65, 1619.20, 1286.81, 1220.23; HRMS (m/z):
C
19H23N4O3 [M + H]+ 355.1770; found, 355.1774; HPLC purity- calculated for C18H20N4O3F [M + H]+ 359.1519; found, 359.1519;
95%; tR ¼ 10.92 min.
HPLC purity-98%; tR ¼ 10.30 min.
4.1.5.5 4-(Hydroxymethyl)-5-(((1-(3-methoxybenzyl)-1H-1,2,3-
4.1.5.9 5-(((1-(3,5-Diuorobenzyl)-1H-1,2,3-triazol-4-yl)
triazol-4-yl)methoxy)methyl)-2-methylpyridin-3-ol (5e). Yellow methoxy)methyl)-4-(hydroxymethyl)-2-methylpyridin-3-ol
(5i).
sticky solid; yield 90%; Rf ¼ 0.4 (DCM/methanol 19 : 1); 1H NMR Yellow sticky solid; yield 96%; Rf ¼ 0.3 (DCM/methanol 19 : 1);
(500 MHz, chloroform-d) d 7.78 (s, 1H, N]CH) d 7.40 (s, 1H, C] 1H NMR (500 MHz, chloroform-d + DMSO-d6) d 7.87 (s, 1H, N]
CH–N) d 7.27 (d, J ¼ 7.6 Hz, 1H, Ar–H) d 6.86 (dd, J ¼ 8.3, 2.5 Hz, CH) d 7.48 (s, 1H, C]CH–N) d 6.85–6.79 (m, 3H, Ar–H), d 5.56 (s,
1H, Ar–H) d 6.80 (d, J ¼ 7.5 Hz, 1H, Ar–H) d 6.74 (d, J ¼ 1.6 Hz, 2H, N–CH2) d 4.91 (s, 2H, CH2–OH) d 4.60 (s, 2H, CH2–O) d 4.52
1H, Ar–H) d 5.42 (s, 2H, N–CH2) d 4.96 (s, 2H, CH2–OH) d 4.46 (s, (s, 2H, CH2–O) d 2.42 (s, 3H, N–C–CH3); 13C NMR (125 MHz,
2H, CH2–O) d 4.43 (s, 2H, CH2–O) d 3.75 (s, 3H, Ar–O–CH3), chloroform-d and DMSO-d6) d 156.20, 149.88, 144.87, 143.80,
d 2.42 (s, 3H, N–C–CH3); 13C NMR (125 MHz, chloroform-d and 135.68, 132.50, 128.31, 115.92, 115.71, 108.78, 72.95, 68.04,
DMSO-d6) d 156.30, 149.66, 144.88, 140.99, 134.97, 127.76, 64.48, 57.54, 23.92; 19F NMR (470 MHz, chloroform-d) d ꢂ103.42
124.99, 118.81, 118.55, 72.91, 68.02, 64.64, 60.09, 58.73, 23.86; to ꢂ103.51 (m); IR (cmꢂ1) n: 3240.35, 2885.91, 2900.62, 2378.85,
IR (cmꢂ1) n: 3248.36, 2932.56, 2873.62, 2375.56, 2248.32, 2378.63, 1622.31, 1278.78, 1223.73; HRMS (m/z): calculated for
1620.20, 1231.80, 1218.36; HRMS (m/z): calculated for
C
C
18H19N4O3F2 [M + H]+ 377.1425; found, 377.1422; HPLC purity-
19H23N4O4 [M + H]+ 371.1719; found, 371.1719; HPLC purity- 100%; tR ¼ 10.88 min.
96%; tR ¼ 10.24 min.
4.1.5.10 5-(((1-(4-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)
methoxy)methyl)-4-(hydroxymethyl)-2-methylpyridin-3-ol
(5f). Yellow sticky solid; yield 85%; Rf ¼ 0.4 (DCM/methanol 19 : 1);
4.1.5.6 5-(((1-(3,5-Dimethoxybenzyl)-1H-1,2,3-triazol-4-yl)
methoxy)methyl)-4-(hydroxymethyl)-2-methylpyridin-3-ol
(5j).
Yellow oil; yield 85%; Rf ¼ 0.4 (DCM/methanol 19 : 1); 1H NMR 1H NMR (500 MHz, chloroform-d) d 7.80 (s, 1H, N]CH) d 7.40
(500 MHz, chloroform-d) d 7.83 (s, 1H, N]CH) d 7.41 (s, 1H, C] (s, 1H, C]CH–N) d 7.32 (d, J ¼ 8.4 Hz, 2H, Ar–H), d 7.16 (d, J ¼
CH–N) d 7.25 (s, 1H, Ar–H) d 6.40 (s, 1H, Ar–H), d 6.36 (s, 1H, Ar– 8.3 Hz, 2H, Ar–H) d 5.43 (s, 2H, N–CH2) d 4.97 (s, 2H, CH2–OH)
H), d 5.39 (s, 2H, N–CH2) d 4.96 (s, 2H, CH2–OH) d 4.50 (s, 2H, d 4.48 (s, 2H, CH2–O) d 4.44 (s, 2H, CH2–O) d 2.43 (s, 3H, N–C–
CH2–O) d 4.44 (s, 2H, CH2–O) d 3.74 (s, 6H, Ar–O–CH3), d 2.44 (s, CH3); 13C NMR (125 MHz, chloroform-d) d 148.53, 145.03,
3H, N–C–CH3); 13C 13C NMR (125 MHz, chloroform-d and 139.96, 135.10, 132.76, 131.63, 129.53, 129.50, 127.57, 122.62,
DMSO-d6) d 161.43, 136.37, 127.44, 122.72, 106.32, 100.48, 68.32, 62.04, 59.72, 53.62, 29.78, 18.65; IR (cmꢂ1) n: 3224.69,
68.30, 61.88, 59.74, 55.54, 54.41, 18.79.; IR (cmꢂ1) n: 3245.63, 2895.81, 2910.25, 2376.68, 2379.36, 1627.91, 1278.70, 1210.35;
2893.60, 2892.36, 2376.78, 2369.32, 1623.20, 1241.81, 1278.23; HRMS (m/z): calculated for C18H20N4O3Cl [M + H]+ 375.1224;
HRMS (m/z): calculated for C20H25N4O5 [M + H]+ 401.1825; found, 375.1222; HPLC purity-97%; tR ¼ 11.56 min.
found, 401.1834; HPLC purity-97%; tR ¼ 10.72 min.
4.1.5.11 5-(((1-(3-Chlorobenzyl)-1H-1,2,3-triazol-4-yl)
methoxy)methyl)-4-(hydroxymethyl)-2-methylpyridin-3-ol
4.1.5.7 5-(((1-(4-Fluorobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
(5k).
methyl)-4-(hydroxymethyl)-2-methylpyridin-3-ol (5g). Brown oil; Yellow oil; yield 88%; Rf ¼ 0.4 (DCM/methanol 19 : 1); 1H NMR
yield 93%; Rf ¼ 0.4 (DCM/methanol 19 : 1); 1H NMR (500 MHz, (500 MHz, chloroform-d) d 7.84 (s, 1H, N]CH) d 7.42 (s, 1H, C]
chloroform-d) d 7.82 (s, 1H, N]CH) d 7.38 (s, 1H, C]CH–N) CH–N) d 7.34–7.27 (m, 2H, Ar–H), d 7.23 (s, 1H, Ar–H) d 7.10 (d, J
d 7.24–7.20 (m, 2H, Ar–H), d 7.03 (t, J ¼ 8.6 Hz, 2H, Ar–H) d 5.44 ¼ 7.2 Hz, 1H) d 5.45 (s, 2H, N–CH2) d 4.99 (s, 2H, CH2–OH) d 4.54
(s, 2H, N–CH2) d 4.98 (s, 2H, CH2–OH) d 4.51 (s, 2H, CH2–O) (s, 2H, CH2–O) d 4.45 (s, 2H, CH2–O) d 2.45 (s, 3H, N–C–CH3);
d 4.44 (s, 2H, CH2–O) d 2.44 (s, 3H, N–C–CH3); 13C NMR (125 13C NMR (125 MHz, chloroform-d) d 151.92, 149.51, 148.78,
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145.10, 130.60, 129.26, 128.25, 126.24, 122.65, 68.43, 61.68,
59.66, 53.67, 18.81; IR (cmꢂ1) n: 3220.69, 2898.81, 2914.38,
2369.82, 2381.41, 1628.85, 1280.01, 1210.56; HRMS (m/z):
4.1.5.16 4-(Hydroxymethyl)-2-methyl-5-(((1-(3-(tri-
uoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
pyridin-3-ol (5p). Yellow oil; yield 87%; Rf ¼ 0.4 (DCM/methanol
19 : 1); 1H NMR (500 MHz, chloroform-d) d 7.82 (s, 1H, N]CH)
d 7.62 (s, 1H, C]CH–N) d 7.51 (d, J ¼ 4.4 Hz, 1H, Ar–H) d 7.49 (d,
J ¼ 7.8 Hz, 1H, Ar–H) d 7.44 (s, 1H, Ar–H) d 7.41 (d, J ¼ 7.7 Hz,
1H, Ar–H) d 5.54 (s, 2H, N–CH2) d 4.98 (s, 2H, CH2–OH) d 4.52 (s,
2H, CH2–O) d 4.46 (s, 2H, CH2–O) d 2.44 (s, 3H, N–C–CH3) 13C
NMR (125 MHz, chloroform-d) d 140.23, 140.11, 135.32, 131.44,
129.95, 125.88, 124.84, 122.68, 68.40, 59.69, 53.74, 29.78; 19F
NMR (470 MHz, chloroform-d) d ꢂ62.64 (s); IR (cmꢂ1) n:
3116.27, 2896.56, 2923.18, 2374.38, 2391.60, 1180.28, 1682.66,
1280.45, 1216.68; HRMS (m/z): calculated for C19H20N4O3F3 [M +
H]+ 409.1488; found, 409.1492; HPLC purity-96%; tR ¼ 8.70 min.
4.1.5.17 4-(Hydroxymethyl)-2-methyl-5-(((1-(2-(tri-
calculated for
C18H20N4O3Cl [M +
H]+ 375.1229; found,
375.1225; HPLC purity-97%; tR ¼ 10.32 min.
4.1.5.12 5-(((1-(3,4-Dichlorobenzyl)-1H-1,2,3-triazol-4-yl)
methoxy)methyl)-4-(hydroxymethyl)-2-methylpyridin-3-ol
(5l).
Yellow oil; yield 92%; Rf ¼ 0.4 (DCM/methanol 19 : 1); 1H NMR
(500 MHz, chloroform-d + DMSO-d6) d 7.75 (s, 1H, N]CH)
d 7.64 (s, 1H, C]CH–N) d 7.24–7.20 (m, 2H, Ar–H), d 7.06 (s, 1H,
Ar–H) d 5.40 (s, 2H, N–CH2) d 4.78 (s, 2H, CH2–OH) d 4.48 (s, 2H,
CH2–O) d 4.38 (s, 2H, CH2–O) d 2.30 (s, 3H, N–C–CH3); 13C NMR
(125 MHz, chloroform-d and DMSO-d6) d 135.45, 131.07, 130.10,
127.62, 123.37, 68.19, 64.43, 59.76, 52.57, 19.27; IR (cmꢂ1) n:
3225.72, 2890.23, 2926.83, 2370.82, 2380.62, 1629.85, 1282.01,
1217.61; HRMS (m/z): calculated for C18H19N4O3Cl2 [M + H]+
410.0912; found, 410.0865; HPLC purity-98%; tR ¼ 10.32 min.
4.1.5.13 5-(((1-(4-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
methyl)-4-(hydroxymethyl)-2-methylpyridin-3-ol (5m). Yellow
sticky solid; yield 91%; Rf ¼ 0.6 (DCM/methanol 19 : 1); 1H NMR
(500 MHz, chloroform-d) d 7.84 (s, 1H, N]CH) d 7.49 (d, J ¼
8.3 Hz, 2H, Ar–H) d 7.48 (s, 1H, C]CH–N) d 7.11 (d, J ¼ 8.3, 2H,
Ar–H) d 5.43 (s, 2H, N–CH2) d 4.98 (s, 2H, CH2–OH) d 4.53 (s, 2H,
CH2–O) d 4.44 (s, 2H, CH2–O) d 2.45 (s, 3H, N–C–CH3) 13C NMR
(125 MHz, chloroform-d) d 148.75, 133.25, 132.48, 129.80,
123.24, 122.56, 68.42, 61.70, 59.64, 53.69, 29.78; IR (cmꢂ1) n:
3226.24, 2892.83, 2924.81, 2374.22, 2386.92, 1685.23, 1280.81,
1216.56; HRMS (m/z): calculated for C18H20N4O3Br [M + H]+
419.0719; found, 419.0715; HPLC purity-99%; tR ¼ 11.91 min.
4.1.5.14 5-(((1-(3-Bromobenzyl)-1H-1,2,3-triazol-4-yl)methoxy)
methyl)-4-(hydroxymethyl)-2-methylpyridin-3-ol (5n). Yellow oil;
yield 91%; Rf ¼ 0.6 (DCM/methanol 19 : 1); 1H NMR (500 MHz,
uoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
pyridin-3-ol (5q). Brown oil; yield 89%; Rf ¼ 0.4 (DCM/methanol
19 : 1); 1H NMR (500 MHz, chloroform-d) d 7.82 (s, 1H, N]CH)
d 7.62 (s, 1H, C]CH–N) d 7.51 (d, J ¼ 4.4 Hz, 1H, Ar–H) d 7.49 (d,
J ¼ 7.8 Hz, 1H, Ar–H) d 7.44 (s, 1H, Ar–H) d 7.40 (d, J ¼ 7.7 Hz,
1H, Ar–H) d 5.54 (s, 2H, N–CH2) d 4.98 (s, 2H, CH2–OH) d 4.52 (s,
2H, CH2–O) d 4.46 (s, 2H, CH2–O) d 2.44 (s, 3H, N–C–CH3); 13
C
NMR (125 MHz, chloroform-d) d 152.85, 132.87, 130.47, 129.06,
126.41, 123.04, 68.43, 61.65, 59.65, 50.39, 19.04; 19F NMR (470
MHz, chloroform-d) d ꢂ58.88 (s); IR (cmꢂ1) n: 3117.83, 2897.75,
2927.20, 2378.42, 2390.56, 1185.75, 1683.20, 1283.30, 1215.78;
HRMS (m/z): calculated for C19H20N4O3F3 [M + H]+ 409.1488;
found, 409.1490; HPLC purity-98%; tR ¼ 12.09 min.
4.2 Biological activity
4.2.1 Inhibition experiment of AChE. For evaluation of
chloroform-d) d 7.85 (s, 1H, N]CH) d 7.48 (s, 1H, C]CH–N) AChE activity of the derivatives, we followed a modied Ell-
d 7.41 (d, J ¼ 8.3 Hz, 2H, Ar–H) d 7.23 (s, 1H, Ar–H) d 7.15 (d, J ¼ man's method using AChE from Electrophorus electricus27.
7.7, 1H, Ar–H) d 5.44 (s, 2H, N–CH2) d 4.98 (s, 2H, CH2–OH) Aer adding 0.1 M phosphate buffer solution (pH-8, 120 mL) to
d 4.53 (s, 2H, CH2–O) d 4.45 (s, 2H, CH2–O) d 2.45 (s, 3H, N–C– every well, AChE enzyme (0.5 U mLꢂ1, 10 mL) along with
CH3); 13C NMR (125 MHz, chloroform-d) d 132.20, 131.15, different concentrations of test compounds (20 mL) were added
130.85, 126.72, 123.30, 117.51, 68.44, 61.74, 59.67, 53.60, 36.88 and incubated at room temperature for 15 minutes. Aer
IR (cmꢂ1) n: 3225.20, 2890.63, 2923.72, 2373.15, 2388.85, incubation, reaction mixture comprising of 75 mM acetylth-
1685.57, 1282.74, 1210.78; HRMS (m/z): calculated for iocholine iodide and 10 mM 5,50-dithiobis-2-nitrobenzoic acid
C
18H20N4O3Br [M + H]+ 419.0719; found, 419.0715; HPLC purity- (DTNB) were added to 96 well plate to generate yellow colour-
95%; tR ¼ 11.79 min.
ation due to 5-thio-2-nitro-benzoic acid. Absorbance was
measured immediately aer addition of the reaction mixture in
a kinetic loop for 10 minutes at 412 nm at an interval of 1
4.1.5.15 4-(Hydroxymethyl)-2-methyl-5-(((1-(4-(tri-
uoromethyl)benzyl)-1H-1,2,3-triazol-4-yl)methoxy)methyl)
pyridin-3-ol (5o). Yellow sticky solid; yield 95%; Rf ¼ 0.4 (DCM/ minute using BioTek Synergy HTX Multimode Microplate
1
methanol 19 : 1); H NMR (500 MHz, chloroform-d) d 7.80 (s, reader. All the samples were assayed in triplicate and IC50 values
1H, N]CH) d 7.62 (d, J ¼ 8.2 Hz, 2H, Ar–H) d 7.43 (s, 1H, C] were determined and expressed as mean ꢁ SEM as generated
CH–N) 7.33 (d, J ¼ 8.1, 2H, Ar–H) d 5.53 (s, 2H, N–CH2) d 4.98 (s, from Graph Pad Prism 8.0 soware.
2H, CH2–OH) d 4.49 (s, 2H, CH2–O) d 4.45 (s, 2H, CH2–O) d 2.43
4.2.2 Antioxidant assay. The antioxidant capacity of all the
(s, 3H, N–C–CH3); 13C NMR (125 MHz, chloroform-d) d 151.97, compounds in our series were measured using oxygen radical
148.67, 145.22, 140.19, 138.21, 131.49, 128.35, 127.46, 126.26, absorbance capacity uorescein (ORAC-FL) method previously
122.75, 68.40, 61.94, 59.72, 53.67, 18.74; 19F NMR (470 MHz, described by Fang Lei et al.28 with some modications. The total
chloroform-d) d ꢂ62.71 (s); IR (cmꢂ1) n: 3115.52, 2895.96, reaction mixture was kept to 200 mL and 75 mM phosphate
2925.63, 2375.38, 2390.58, 1182.06, 1683.57, 1281.54, 1213.61; buffer solution was used for every assay. In dark 96 well plate, 25
HRMS (m/z): calculated for C19H20N4O3F3 [M + H]+ 409.1488; mL of sample (8 mM)/Trolox (8 mM)/buffer was added along with
found, 409.1489; HPLC purity-97%; tR ¼ 12.57 min.
150 nM uorescein solution (150 mL) and incubated in dark at
37 ꢀC for 30 minutes. Following this, free radical initiator,
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12 mM AAPH (25 mL) was added and immediately aer addition,
uorescence readings were taken at excitation 485 nm and
emission at 535 nm in a kinetic loop for 15 minutes at 1 minute
interval. All the samples were assayed in triplicate in BioTek
Synergy HTX Multimode Microplate reader. The area under the
uorescence decay curve (AUC) was calculated for every sample
and total antioxidant capacity of the compound was determined
by substractingit's AUC from the AUC of the blank. ORAC-FL
values are expressed as Trolox equivalents and ORAC-FL value
of Trolox is taken as 1. The ORAC-FL values of three individual
experiments were expressed as expressed as mean ꢁ SEM as
generated from Graph Pad Prism 8.0 soware.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
Tiyas Pal and Saipriyanka Bhimaneni are thankful to Depart-
ment of Pharmaceutical, Ministry of Chemicals and Fertilizers
for providing nancial assistance. Spectral study was carried
out at Central Instrumental Facility NIPER-R. NIPER-R/
Communication/123.
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