Synthesis, molecular docking and spectroscopic studies of pyridoxine carbamates as metal chelator

Article abstract of DOI:10.1016/j.molstruc.2020.128837

Herein, we have reported synthesis, characterization, molecular docking and metal chelating potential of various pyridoxine carbamates for biometals (copper, zinc and iron) via spectroscopic methods. All the derivatives showed metal chelation ability and 4g was found the most potent metal chelator, having a binding stoichiometry of 1:1 with Cu⁺² ion. Interactions of metal with ligand 4g was studied and the participation of both carbamate and free –OH group in complex formation was confirmed. All the synthesized compounds showed druglikeness properties, passing the lipinki's Rule of five. According to Pass study 4a, 4b, 4d and 4h to show significant nootropic activity (Pa > 0.60) in comparison to donepezil. Fluorescence quenching study and analysis with BSA with 4g showed static quenching mechanism. Molecular docking showed probable site of binding with least binding energy of ?5.9 kcal mol^?1. The compound 4g also showed binding with acetylcholinesterase in UV–Vis spectrum. The study concludes that pyridoxine-carbamates are biometal chelator, possess other properties also like drug likeness, binding with BSA and acetylcholinetersae.

Full text of DOI:10.1016/j.molstruc.2020.128837

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Synthesis, in silico, molecular docking and spectroscopic studies of pyridoxine
carbamates as metal chelator
Tiyas Pal, Pooja Patil, Abha Sharma
PII:
S0022-2860(20)31162-5
DOI:
https://doi.org/10.1016/j.molstruc.2020.128837
MOLSTR 128837
Reference:
To appear in: Journal of Molecular Structure
Received Date: 22 February 2020
Revised Date: 5 June 2020
Accepted Date: 5 July 2020
Please cite this article as: T. Pal, P. Patil, A. Sharma, Synthesis, in silico, molecular docking and
spectroscopic studies of pyridoxine carbamates as metal chelator, Journal of Molecular Structure (2020),
doi: https://doi.org/10.1016/j.molstruc.2020.128837.
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Credit Author Statement
Tiyas Pal: In silico and spectroscopic study
Pooja Patil: Synthesis and characterization of molecules
Abha Sharma: Design and investigation of study and writing the manuscript

Synthesis, In Silico, Molecular Docking and Spectroscopic Studies of Pyridoxine
Carbamates as Metal Chelator
Tiyas Pal, Pooja Patil, and Abha Sharma*
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research,
Raebareli (NIPER-R), India
*E-mail: abha.sharma@niperraebareli.edu.in; abha_052002@yahoo.co.in
Abstract
Herein, we have reported synthesis, characterization, molecular docking and metal chelating potential of
various pyridoxine carbamates for biometals (copper, zinc and iron) via spectroscopic methods. All the
derivatives showed metal chelation ability and 4g was found the most potent metal chelator, having a
+
2
binding stoichiometry of 1:1 with Cu ion. Interactions of metal with ligand 4g was studied and the
participation of both carbamate and free –OH group in complex formation was confirmed. All the
synthesized compounds showed druglikeness properties, passing the lipinki’s Rule of five. According to
Pass study 4a, 4b, 4d and 4h to show significant nootropic activity (Pa > 0.60) in comparison to donepezil.
Fluorescence quenching study and analysis with BSA with 4g showed static quenching mechanism.
-1
Molecular docking showed probable site of binding with least binding energy of -5.9kcal mol . The
compound 4g also showed binding with acetylcholinesterase in UV-Vis spectrum. The study concludes that
pyridoxine-carbamates are biometal chelator, possess other properties aalso like drug likeness, binding with
BSA and acetylcholinetersae.

1. Introduction
Metal homeostasis in brain is very stringent in normal physiological conditions for proper functioning of the
neuronal tissues. Certain storage proteins like transferrin, ceruloplasmin sequester metals and maintain
normal levels of bio-metals. There are diseases in which metal dyshomeostasis is involved in pathogenesis
for instance, Alzheimer’s disease (AD). AD is one of the most prominent occurring neurodegenerative
diseases affecting mostly the population above the age of 60. However, the exact pathophysiology behind
the occurrence of the disease is still unknown and metal dyshomeostasis is one of the known etiology of AD
[
[1, 2]. Patients with AD have abnormal burden of bio-metals, mainly Fe, Cu and Zn in the subcortical
regions of amygdala, hippocampus and olfactory bulb along with the neocortical region, might contributes in
degeneration of neurons[3]. Several studies have shown high metal deposits in the neurofibrillary tangles
(NFT)formed due to hyperphosphorylated tau protein in AD brains of mice[2, 4, 5]. Aβ is a metalloprotein
with binding sites for transition metals (Fe,Cu,Zn) comprising of both high and low affinity ones. These bio-
metals bind to the metalloprotein and get reduced due to the electron donation by methionine. As a result of
which, H O is produced. H O serves as a substrate for Fenton reaction liberates OH radical and which in
2
2
2
2
turn is responsible for all oxidation reactions like lipid peroxidation, oxidation of proteins and nucleic acids
and enhancing oxidative stress. H O has been also found to have reacted with the reduced metals which
2
2
bind to Aβ, stabilizes neurotoxic Aβ by forming cross linking oligomers[6-9]. The impairment of bio-
4
2
metals can be attenuated by using metal chelators. The protective effect of metals chelators has been
investigated in several diseases including Wilson’s, Thalassemia, multiple sclerosis and AD. There are some
drugs which are used as chelators in metal toxicity like penicillamine as in the case of copper, lead, mercury
and nickel. Deferoxamine is known to treat iron toxicity and tested for AD as well [10]. Taking up the
restoration of metal ion homeostasis as a challenge, there are reports of other molecules being tested against
AD, given in Figure 1[11].The accumulation of metal ions in many neurodegenerative diseases such as iron
in Parkinson’s and copper, iron & zinc in AD have been investigated for developing therapeutic targeting to
reduce the involved metal ions[12-15]. After the several ramifications of the drugs targeting Aβ cascade
hypothesis, metal dyshomeostasis is one such area which remains partly excavated in AD[13, 16, 17].
Therefore, disease progression can be curbed by using chelating agents which can form complex with the
transition metals, targeting metal dyshomeostasis hypothesis.
Vitamin B exist in three forms and its active form is pyridoxal 5′-phosphate that acts as coenzyme in some
6
enzymatic metabolism offat,carbohydrates and amino acid[18]. It has been also discloses that pyridoxine
serve as potent antioxidant via inhibiting free radicals production[19]. Deficiency of pyridoxine also leads to
elevated level of homocysteine (Hcy) that is correlated to increased Aβ levels ant deposition of it leads to
developing AD as indicated by epidemiological and clinical studies [20]. Therefore, Hcy level could be
maintained by taking pyridoxine supplement that may be helpful in AD treatment [21, 22].Bovine serum

albumin (BSA) is homologous to human serum albumin (HSA) is a main protein present in plasma and act
as transporter of drug. Since transportation of drug to the target site remains very crucial for desired activity,
we performed spectroscopic studies of binding affinity for serum protein by taking the most potent chelator
among our series as the probe, henceforth BSA study is often exploited extensively for determining binding
mechanism of drug molecules[23, 24].
i
Cl
O
HS
N
OH
O
N
I
NH2
OH
OH
D-Penicillamine
Clioquinol
Deferiprone
ii
iii
iv
Figure 1: (a) Chelator as drug (i-iii) (b) Metal chelator tested in AD (iii, iv)
2
2
. Results and Discussion
.1 Design of Pyridoxine Carbamate
There are several functional groups such as hydrazones, semicarbazone, amide, vicinal hydroxyl groups
involved in metal chelation[25, 26]. Pyridoxine vicinal hydroxyl chelate metal ions as investigated by NMR
studied[27]. We have reserved these groups in pyridoxine and introduced carbamate moiety with the
assumption ofC=O and the adjacent N-H group of carbamatemay potentiate the metal chelation activity as
shown in figure 3.The amide-ester hybrid properties of carbamates protects it from enzyme hydrolysis and
renders chemical stability to it [28].The carbamate functionality interacts with other molecules via hydrogen
bonding whichinvolve carboxyl and -NH groups. Modulation in biological properties, stability and
pharmacokinetic profile can be obtained by substitution around the carbamate functional group. The target
molecule has been designed so that the metal ion can bind to more than one site and that it follows the
Pearson’s principle of “hard and soft acids and bases”. The transition bio-metals whose abnormal
accumulation is held responsible for the pathophysiology of AD are copper, iron and zinc. The mentioned
bio-metals require intermediate hard and soft acids and bases for metal chelation which must comprise of N
and O atoms[29]. The designed molecule has been hypothesized to act as a metal chelator shown in figure 2.

Figure 2: Metal chelation atoms are shown in blue colour
2
.2 Chemistry
The synthetic method for synthesis of derivatives of pyridoxine carbamates is shown in Scheme
.Commercial pyridoxine hydrochloride (1) was used as starting material which was reacted with acetone in
the presence of concentrated sulphuric acid to give 3,4’O-isopropylidenepyridoxine (2). In the next step,
,4’O-isopropylidenepyridoxine was refluxed with acetonitrile in the presence of substituted isocyanates,
1
3
resulting in compounds 3(a-j). The scheme was concluded by deprotection of the acetonide ring of 3,4’O-
isopropylidenepyridoxine of compounds 3(a-j), in the presence of 10% hydrochloric acid and
tetrahydrofuran under nitrogen atmosphere and was refluxed for about 6-11 hours resulted to final
1
13
compounds 4(a-j). Structures of all target compounds were characterized by HNMR, C NMR, ESI-MS,
and HRMS.
Scheme1: Synthesis of compounds 4a-j. Reagents and conditions:i) conc. H SO , acetone, rt, 6h; ii) RNCO,
2
4
acetonitrile, reflux; 4-8h; iii) 10% HCl, THF, N atmosphere, 70˚C, 6-11h.
2
Table 1: Derivatives of Pyridoxine -Carbamates Synthesized

a
Compound
R
M.P (˚C)
Yield(%)
95
4a
152-155
154-156
169-170
175-179
148-150
167-170
164-166
4
b
94
96
92
91
94
91
4
c
4d
4
e
4
f
4g
4
h
134-138
142-145
92
94
4
i
4j
157-162
93
a
M.P.= melting point
.2 Metal Chelation study using UV-Visible Spectrophotometer
2
The series of compounds 4(a-j)were checked for metal chelation ability within the wavelength range of 200-
00nm by using of bio-metal salts-copper(II) chloride, zinc sulphate, and iron (III) sulphate. Metal chelation
was observed at incubation periods of 1minute, 10minutes and 24 hours; 10 minutes proved to be the
6
2
+,
2+
3+
optimum incubation period. These salts generated metal ions (Cu Zn and Fe ) in solution which were
found in abundance in the brains of AD patients. To test for metal chelating activity, the synthesized
2
+,
2+
3+
compounds 4(a-j) separately were added to the solution containing metal ion (Cu Zn and Fe ) and
scanned at wavelength range 200-600nm. All the compounds showed significant reduction in absorbance.
Compound 4gshowed maximum metal chelation while compound 4d showed the least. Compound 4g
showed selectivity towards copper, with marginal difference with the other bio-metals studied. Any changes
in absorbance band related to its intensity and wavelength indicates metal chelation as reported in pyridoxal
phosphate metal chelation study. Pyridoxal phosphate formed metal-ligand complexesand showeda decrease
in absorbance as well as shifts in the absorption spectrum compared to pyridoxal phosphate alone, these
changes confirmed metal chelation[30].

All the pyridoxine carbamates exhibited metal chelation with remarkable reduction in absorbance upon
addition of transition metals. The presence of different substituents caused changes in the electronic
arrangement which was revealed by the excitation spectra of the individual compounds. As shown in Figure
4
,pyridoxine itself shows three λmax at 256nm,290nm and 325nm. On protection of the –OH groups at 3,4
positions, only one λmaxat 280nm was observed; and with the introduction of the carbamate moiety with
protected pyridoxine yielded peaks at 245nm and 284nm. On deprotection of the acetonide ring, compound
4
g showed three peaks at 245nm, 288nm and 325nm.Since 4g showed the best metal chelating ability, it’
ability was compared to that of pyridoxine. Interestingly, it was found that all the three peaks of pyridoxine
participated in metal chelation like that of 4g,though was not as proficient as 4g. There was more than three
folds spurt in ability to chelate metals at λmax 325nm, as shown in Figure 5. Pyridoxine alone showed 21.7%
of metal chelation at 325nm,while, with the introduction of carbamate moiety with a trifluoromethyl
substituent showed 76.92% of it’s activity.The ten derivatives showed different affinities towards the bio-
metals, thereby generating difference in their ability to chelate metals due to the presence of different
substituents. However, the presence of an electron withdrawing group or electron donating group influenced
the extent of metal chelation. The presence of bromine and chlorine group in the para position showed only
marginal difference of 2% in metal chelation properties, proving the later to be the better one. However, the
presence of electron donating groups like methoxy in the para position, showed 7% lesser ability to chelate
metals than chlorine, which also contributes towards donating electrons. Presence of electron withdrawing
groups in the para position like -NO did not show any alteration in metal chelation properties. Difference in
2
activity was also checked with di-substituted ring. Presence of chlorine in the para position and
trifluoromethyl in the meta position exhibited 76.92% metal chelation proving to be the best among all in the
series. Further investigation was carried out by varying the chain length from carbamate moiety. The
derivative with 3 carbon chain lengths showed 50% metal chelation but the compound with 4 carbon chain
length showed 48% activity. Figure 3 shows UV-vis spectra of compounds 4(a-g).
To determine the stoichiometry of the compound 4gin complex with copper (chelated the most), mole ratio
method was used. Increasing concentration of copper bio-metal was used (1.5-90µM), keeping the
compound concentration fixed. The absorbance change was recorded at 245nm and this change in
absorbance was plotted against molar ratio. It was observed that there was a sharp decline in absorbance
followed by a steep increase resulting in two straight lines. These two straight lines intersected at mole
fraction of 0.5, and hence it can be deduced that the metal complex stoichiometry exhibits a binding of 1:1
2
+
ratio of 4g-Cu complex (Figure 6)[31].

Figure 3: The UV spectra of metal chelation of pyridoxine - carbamates4(a-j)after 10minutes incubation

Figure 4: Excitation spectra of Pyridoxine, Compound 2,3g and 4g
Figure 5: Differences in extent of metal chelation in pyridoxine and compound 4g

2
+
Figure 6: Determination of stoichiometry of 4g-Cu
2
.3 Metal Chelation study using Fluorescence Spectrophotometer
To further confirm of the metal chelation activity of the compounds, all the synthesized compounds were
studied for fluorescence activity. All the derivatives showedsharp fluorescent emission intensity around 390-
4
90nm. After formation of ligand-metal complex, fluorescence was observed. The ligand served as the
fluorophore in the metal-ligand complexes formed. Three, out of ten compounds (4c,4d, 4h) exhibited shifts
in fluorescence band towards shorter wavelength also known as hypsochromic shift. The metal-ligand
complex of Cu(II)-4c showed both hyperchromic and hypsochromic shifts, Zn(II)-4c and Fe(III)-4c
manifested fluorescence quenching and hypsochromic shifts. All the metal-ligand complexes showed
disappearance of the curve in the spectra at 350nm, which free 4c emission spectrum had. In the case of 4d,
shifts were observedafter one hour ofincubation. Presence of non-bonding electrons of oxygen atom may
serve as electron donor to the transition metal ion forming complex, therebyexhibiting a shift towards shorter
wavelength. Replacement of aromatic substituent with allylic system as in the case of 4h showed us slight
different spectra. Unlike 4c and 4d, 4hlacked any hyperchromic shifts in the metal complexes. However,
hypsochromic shift with only quenching was observed in case of 4h.This compound complex also
manifested a disappearance of curve at 338nm, which the free 4h spectrum showed (Table 1).Extent of
chelation by individual compounds withbio-metalswas found to be the same as observed on UV-visible
spectrophotometer.
The rest of the compounds showed only hypochromic shifts with no blue or red shifts. Reduction in
fluorescence intensity was probably due to the fact that less electrons were available to the phenyl ring in the
metal-ligand complex formed[32]. Compound 4g was found to be the best among all and showed selectivity
towards copper with a decline in fluorescent intensity of 66%. Nodifference was observed in the emission
spectra with variation in chain length with the phenyl substituents (4i,4j). Changing the position of chlorine
(4a, 4b) did not exhibit any subtle changes in emission intensity of the spectra. 4a chelated zinc and iron the

most while copper the least, while 4b chelated copper and zinc the least and iron the most. Thus, by
changing the substitution of chlorine at the meta position, the strength of chelation towards zinc in
comparison to 4a reduced (Figure 7).
Table 2: Effect of metal on fluorescence band of compound
λmaxof
intensity of ligand
nm)
emission
λmax of emission intensity of
% change in emission
intensity
Ligand
∆λmax
metal-ligand complex
(
Cu-4c
Fe-4c
Zn-4c
Cu-4d
Fe-4d
Zn-4d
Cu-4h
Fe-4h
Zn-4h
390
391
393
398
394
398
389
388
388
4.39
3.52
1.76
2.05
5.79
2.05
4
3.65
6.8
4
c
395
400
393
36.17
7.18
4
4
d
h
2.05
5.51
34.75
29.46
29.46
5
5

Figure 7: Fluorescence spectra of pyridoxine-carbamates4(a-j) after 10 minutes incubation

2
.4 IR Spectroscopic Study of Copper (II) - 4g Complex
After screening the compounds for metal chelation activity, 4g proved to be the best among the series and
was found to chelate copper (II) best. Interactions between Compound 4g and copper (II) were studied with
the help of IR Spectroscopy. A complex was formed between 4g-Cu(II) which got precipitated out and the
interactions were studied using IR (Figure 8). The IR spectrum of 4g- Cu(II) complex showed distinctive
-1
changes in the region 1460-750cm when compared with the spectrum of the compound 4g alone.
-1
-1
Interactions of 4g with metal caused diminishing of C=O stretch and a shiftat1741.68cm to 1739.44 cm ;
which can be an effect due to M L electron donation. A complete disappearance of C-O stretch at 1168.31
-1
cm was observed in the spectrum of the complex. The C=C aromatic stretch also showed a marked shift
-1
-1
from 1537.75cm to 1518.13cm . All the observations drawn from the differences in spectra confers that
the carbamate moiety participates in complex formation with Cu(II). The sharp free –OH peak observed of
-1
-1
pyridoxine at 3740.13cm undergoes a higher shift at 3742.32cm and became sharper. The complex yielded
-1
an additional broad peak at 3346.81cm , which may be due to the participation of -OH in metal chelation.
a
b
Figure 8- (a) IR spectrum of 4g (b) IR spectrum of 4g- Cu(II) complex
2
.5 ADME Property Prediction
We have assessedpotential drug-like properties of all derivative 4a-4j. We subjected all novel pyridoxine
analogues to “Lipinski’s Rule of Five” which predicts the druglikeness behaviour of the compounds (Table
1
).Besides the known drug Donepezil, all the compounds were found to obey the “Lipinski’s Rule of Five”.
For treatment of AD, it is very vital to detect the BBB crossing ability of the compounds, henceforth,
physicochemical properties, like topological polar surface area (tPSA) ,cLogP, and LogBB (the logarithm of
brain to blood concentration ratio) were considered. These aforesaid parameters are known to be reliable
evidences

for BBB penetration. Examinations have confirmed that the available CNS drugs in the market, were
considered successful agents having a tPSA value less than 90 Å,cLogP value between 2−5 and a LogBBB
value greater than −1. As shown in Table 2, Donepezil and all the pyridoxine carbamates, except 4d and 4e,
have cLogP, TPSA and LogBBB values within or close to the desirable range for all parameters. For4d and
4
e, the cLogP, value is within the allowed range, but the LogBBB (-1.82 and -1.21 respectively) and tPSA
value (142.96 and 100.38 respectively) increased dramatically relative to the other pyridoxine analogues due
to the presence of –NO and –OCH groups. In summary, most of the novel pyridoxine carbamates
2
3
demonstrate appropriate physicochemical characteristics that suggest their druglikeness and facilitates their
BBB permeation to act as orally-available drugs for the CNS.
Table 3: Calculated physiochemical properties of Donepezil and pyridoxine carbamates
HBA
HBD
Rot.
a
a
a
a
c
Sr.No. Compd.
M.W
LogP
tPSA
cLogP
LogBBB
b
(N+O) (NH+OH) bonds
1
2
3
4
5
6
7
8
9
1
169.18
322.75
322.75
367.20
333.30
318.33
336.77
390.74
252.27
-0.14
2.22
2.22
2.49
1.32
1.54
2.71
3.14
0.69
4
6
6
6
9
7
6
6
6
3
3
3
3
3
3
3
3
3
2
5
5
5
6
6
5
6
6
73.05
91.15
91.15
91.15
-0.345
1.713
1.713
1.863
-0.99
-0.95
-0.95
-0.92
-1.82
-1.21
-0.87
-0.81
-1.00
4a
4b
4c
4d
4e
4f
4g
4h
142.96 0.983
100.38 0.8365
91.15
91.15
91.15
2.212
2.627
0.935
1
1
0
1
4i
4j
330.38
344.41
2.43
2.85
6
6
3
3
8
9
91.15
91.15
2.637
3.166
-1.01
-0.72
Donep-
ezil
13
379.50
4.01
4
0
6
38.77
4.60
0.26
Reruie
red
-
-
≤500
≤5
≤10
≤5
≤10
90
2-5
>-1
Param-
d
eters
a
Calculated using ChemBioDraw Ultra14.0.
b
Calculated using moleinspirationcheminformatics(http://www.molinspiration.com/).
c
LogBB = −0.0148 × TPSA + 0.152 × cLogP + 0.139(18).
d
Required parameters necessary to fulfill appropriate physiochemical properties as judged appropriate
according to Lipinski’s Rules and those important for BBB permeation[33, 34].

2
.6 PASS Prediction
The PASS prediction results of pyridoxine carbamates series 4a-4j for nootropic activity, relevant
toneurological disorders are listed in Table 2. The probability of the pyridoxine carbamates series for being
active with Pa values greater than 0.50, considered as potent nootropic. Among the tested compounds 4a, 4b,
4
4
d and
hhave shown significant nootropic activity (Pa > 0.60) incomparison to donepezil (Table 3). This predicted
nootropic activity for synthesized compoundsdirectly correlates with the memory and learning aspect of AD
and can be considered forfurther biological evaluation[35].
Table 4:Probable activities predicted for the selected compounds using PASS server
Sr.No.
Compound
Pa
Pi
1
2
3
4
5
6
7
8
9
4a
0.517
0.529
0.496
0.507
0.498
0.361
0.390
0.508
0.610
0.670
0.561
0.120
0.113
0.134
0.126
0.132
0.257
0.226
0.125
0.072
0.049
0.040
4b
4c
4d
4e
4f
4g
4h
4i
4j
1
1
0
1
Donepezil
2
.7Experimental log p value of 4g
Experimental logp value was found out for the compound 4g. A calibration curve (Figure 9) was
constructed taking increasing concentration of compound within the range 20-120µM. The log P value was
found 2.12 by the formula log P=concentration of 4g in 1-octanol/concentration of 4g water. The log p range
to cross the BBB is 1.5-2.7, and thus 4gwas found lipophilic enough to cross the BBB.

1
.2
1
y = 0.0088x - 0.0095
R² = 0.9979
0
0
0
0
.8
.6
.4
.2
0
0
20
40
60
80
100
120
140
Concentration(µM) of 4g
Figure 9: Determination of log P value of 4g
.8 Fluorescence study of BSA
2
BSA is considered as one of the most crucial protein which is involved in transportation of drugs to the
desired target. It consist of 583 amino acids with tryptophan in it’s hydrophobic cavity[24]. An experiment
of fluorescence quenching was performed with BSA and with chelator molecule 4g.Fluorescence of BSA is
due to the presence of amino acids tryptophan, phenylalanine and tyrosine. Fluorescence quenching refers to
decrease in fluorescent intensity and in BSA, fluorescence quenching occurs due to presence of Trp-213 and
Trp-134 of the hydrophobic pocket[36]. Changes in fluorescence emission intensity are observed due to the
conformational changes of tryptophan due to substrate binding. In our case, the substrate caused
conformational changes in the protein molecule due to the binding of compound 4g. A strong fluorescence
emission peak was observed at 343nm after being excited at 277nm. On increasing concentration of 4g,
BSA-4g complex was formed which caused quenching (Figure 10A). The data was further analysed using
Stern –Volmer equation (Eq. (1))[37]:
F /F= 1 + Kqτ [Q] =1+ K [Q]
(1)
0
0
SV
F0 and F are the fluorescence intensities of BSA in the absenceand presence of the quencher, respectively.
Kq is the bimolecular quenching rateconstant of the biomolecule, KSV is the Stern–Volmer
-8
dynamicquenching constant, τ is the average lifetime of the biomolecule without quencher (τ = 10 s)[37],
0
0
and [Q] is the concentrationof quencher, in our case 4g (Figure 10B). Henceforth, from equation 1, it can be
said that ,
kq= K / τ (2)
SV
0
After analysing the data, the findings were summed up in Table 3.
Table 5: Stern−Volmer Quenching Constant (K ) andBimolecular Quenching Constant (kq) of the
SV
Complex 4g-BSA
-
1 -1
-1
a
Probe
Temperature
310K
kq (M s )
K (M )
R
SV
1
3
5
4
g
1.13 X 10
1.13 X 10
0.99

a
represents the regression coefficient
1
0
−1 −1
Scientific reports shows that the maximum scatters collision kq is 2.0 × 10 L mol s .(8)The data found
from the Stern- Volmer plot confirms the fluorescence quenching is not the result of dynamic collision
quenching, rather a consequenceof static quenching. Static quenching process is a result of formation of
stable complex between the probe and the protein. On the contrary, dynamic quenching is a consequence of
collisional encounters between the protein and the probe[36].
Double log plot for determination of binding sites and binding constants was plotted.Taking equation 3,
F0
F
ꢁ
log ꢀ
ꢂ = log K + n log[ꢃꢄ]
(3)
b
F
K and n from equation 3 are the binding constant and the number of binding sites. After analysis, K was
b
b
5
-1
found4.98 X 10 M and the number of binding sites was found out to be 0.25 (Figure 10C).
1
1
1
1
.65
A
B
1.6
.55
1.5
.45
1.4
.35
0
0.5
1
1.5
2
2.5
3
-6
[
4g] X 10 M

-
-
-
-
0.15
C
-
0.2
0.25
-
0.3
0.35
-
0.4
0.45
-
0.8
-0.6
-0.4
-0.2
0
0.2
0.4
0.6
-6
log[4g] x 10
Figure 10: (A) Fluorescence spectra of BSA (2.5 μM) with varying concentration of 4g (0.25-2.5µM). (B)
Stern−Volmer plots for fluorescence quenching of BSA−4g complex. (C) Double log plot of BSA-
4
gcomplexforthe determination of the number of binding sites and the binding constant value
2
.9 Molecular Docking Results
Molecular Docking study was carried out to anticipate the probable binding location of the compound4g in
the microenvironment of the tryptophan residue(s) of the serum albumin; the binding position of 4g is shown
in Figure 11a. The crystal structure analysis has revealed that the protein comprise of three binding sites I, II
and III with subdivision to A and B in each binding site. Molecules are reported to have bound to either site I
or site II of the BSA molecule[38, 39]. (22) Docking studies have revealed that the molecule 4g has possible
hydrogen interaction with Asp-111, Glu-424, Pro-420 of BSA (Figure 11b). In addition to this, 4gis
observed to have surrounded with amino acids Pro110,Thr518, Leu112 which might play an important role
in stabilizing the binding of 4g with BSA through some electrostatic interactions. The optimized docking
-1
model gave the least binding energy of -5.9kcal mol . However, distance from the hydrophobic Trp213 and
Trp134 residue was found to be 26.4 Å and 20.3Å. Docking results cannot infer whether the binding of the
4
g molecule to BSA can cause conformation changes in the Trp213 and Trp134 residue as the binding
location of the 4g was not in close proximity to that of the afore stated residues.However, quenching studies
with BSA validated that binding of the 4g to the protein caused conformational changes in the tryptophan
residues that led to decrease in fluorescence intensity.

1
1a
1
1b
ii
i
iii
Figure 11: (11a) Docking poses of 4g with BSA. (11b) H-bonding interactions of BSA residues with 4g (i)
H-bonding with ASP-111(ii) H-bonding with PRO-420 (iii) H-bonding with GLU-424.
2
.10 In-vitroAChE binding study with UV-Visible spectrophotometer
Cholinergic hypothesis has been the most studied hypothesis in AD. Therefore, cholinergic crisis in brain
makes the acetylcholinesterase a very vital enzyme in the pathogenesis of AD. So,we decided to study the
in-vitrobinding of the compound4g with AChE enzyme. UV-Visiblespectrum was obtained within the
wavelength 200-600nm. The concentration of the compound 4g was kept constant (60µM) and the reaction
mixture prepared with 1:1 combination of compound and enzyme. After an incubation of 30 seconds, the
spectrum was recorded. There was a characteristic downward binding curve obtained at 245nmwith no

bathochromic or hypsochromic shifts. Therefore, it can be confirmed that the 4gis binding with the enzyme
causing change in intensity of 4g absorption band (Figure 12).
1.2
1
0.8
0.6
0.4
0.2
0
4
g
4g+Enzyme
2
20
240
260
280
300
320
340
360
380
Wavelength(nm)
Figure 12: UV-Visible spectra of Enzyme complex with 4g and of 4g alone
3
3
. Materials and Methods
.1 Materials
All chemical reagents and solvents were purchased from Sigma Aldrich and SpectrochemPvt.Ltd. and used
without further purification. Reactions were monitored by Thin Layer Chromatography (TLC)using Merck
silica gel 60 F254 precoated plates (0.25mm)and the compounds were examined under UV chamber
(Ultraviolet fluorescence analysis cabinet-PSI).Column chromatography was performed with silica gel (100-
1
13
1
2
00 mesh). All products were characterized by H NMR, C NMR, IR Spectroscopy and ESI-MS. H and
C NMR were recorded on BRUKER DRX 300 MHz and 75 MHz respectively with chemical shift
1
3
expressed in parts per million in DMSO-d6 at 27°C downfield from TMS as an internal standard and
coupling in Hertz (Hz). Splitting patterns are described as singlet (s), doublet (d), triplet (t) and multiplet
(m). For 13C NMR reference for DMSO-d6 appeared at 39.51 ppm. Electron spray ionization mass spectra
were recorded on JEOL JMS-600H high resolution spectrometer using EI mode at 70 eV. IR spectra were
recorded on a BRUKER ECO-ATR Spectrometer. Melting points were recorded on labindia melting point
apparatus and are uncorrected.
Methods:

General procedure for the protection of (5-hydroxy-6-methylpyridine-3,4-diyl)dimethanol (1):
To a suspension of pyridoxine hydrochloride (1) (2.00 g, 9.75 mmol) in acetone (40 mL) was added
concentrated sulfuric acid (4 mL) dropwise at room temperature. The mixture was stirred for 24 h.
Completion of the reaction was monitored by TLC. The reaction mixture was neutralized with aqueous
sodium bicarbonate, concentrated under reduced pressure, and extracted with dichloromethane (40 mL×3).
The organic phase was washed with brine and dried over anhydrous Na SO . The DCM layer was
2
4
concentrated in vacuum to provide a crude product, which was recrystallized from ethyl acetate to give
compound (2) as white crystals.
(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)methanol (2).White solid (65%; 1.33 g); melting range
˚
-1
1
=
108.0-109.0 C; R = 0.46 (hexane-EtOAc = 2/8 v/v); FT-IR (ATR) νcm : 3619, 2960, 2840, 1524; H
f
13
NMR (300 MHz; CDCl ; TMS): δ 7.89 (s, 1 H), 4.94 (s, 2 H), 4.57 (s, 2 H), 2.39 (s, 3 H), 1.55 (s, 6 H); C
3
NMR (75 MHz; DMSO; TMS) δ 145.44, 145.09, 138.42, 130.59, 125.24, 99.41, 58.24, 57.88, 24.53, 18.24;
+
ESI-MS (m/z): calcd for C H NO [M + H] 209.25; found, 210.3.
8
11
3
General procedure for synthesis of Carbamates (3a-j):
A mixture of (2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)methanol (2) (0.2 gm, 0.95 mmol) and
desired isocyanate (0.95-0.97 mmol ) in acetonitrile (3 mL) was refluxed for 6-9 h. The duration of the
reaction was variable depending upon the nature of isocyanate used. The reaction mixture was concentrated
under vacuum and then distilled water (15 mL) was added followed by the extraction with EtOAc (15
mL×3). The organic phase was washed with brine and dried over anhydrous Na SO . The ethylacetate layer
2
4
was concentrated under vacuum to afford the crude product, which was purified by column on silica using
0:80 ratio of EtOAc-hexane solvent system to provide compounds 3a-jin 64-80 % yield.
2
(2,2,8-trimethyl-4H-[1,3]dioxino[4,5-c]pyridin-5-yl)methyl (3-chlorophenyl)carbamate(3a) White solid
˚
-1
(
68%; 0.222 g); melting range = 195.0-197.0 C; R = 0.52 (hexane-EtOAc = 6/4 v/v); FT-IR (ATR) νcm :
f
1
3
7
309, 2951, 2814, 1701, 1524, 1220, 763; H NMR (300 MHz; DMSO; TMS): δ 9.94 (s, 1 H), 8.04 (s, 1 H),
.59 (t, J=1.89 Hz, 1 H), 7.27 - 7.40 (m, 2 H), 7.02 - 7.08 (m, 1 H),5.11 (s, 2 H),4.97 (s, 2 H), 2.37 (s, 3
+
H),1.51 (s, 6 H); ESI-MS (m/z): calculated for C H ClN O [M + H] 362.81; found, 363.3.
1
8
19
2
4
General procedure for deprotection of Compounds (3a-j):
The corresponding compounds 3a-j (0.60-0.65 mmol) was dissolved in THF (5 mL), and 10% HCl solution
(4 mL) was added. The reaction mixture was refluxed for 6-11 h under an argon atmosphere. After complete
reaction, THF was evaporated under reduced pressure. The residue was neutralized with aqueous saturated
sodium bicarbonate sloution, and extracted with EtOAc (20 mL×3). The organic phase was washed with
brine and dried over anhydrous Na SO . The solvent was evaporated to dryness under reduced pressure to
2
4

provide a crude product, which was purified on a silica chromatography using 40:60 ratio of EtOAc-hexane
solvent system afford compounds 4(a-j).
(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-yl)methyl(3-chlorophenyl)carbamate (4a)Light brownish
˚
solid (95%; 0.188 g); melting range = 152.0-155.0 C; R = 0.45 (hexane-EtOAc = 4/6 v/v); FT-IR (ATR)
f
-1
1
νcm : 3619, 3309, 2952, 2821, 1702, 1528, 1219, 760; H NMR (300 MHz; DMSO; TMS): δ 9.93 (s, 1 H),
7
2
1
+
.99 (s, 1 H), 7.60 (t, J=1.79 Hz, 1 H), 7.26 - 7.41 (m, 2 H), 7.01 - 7.08 (m, 1 H), 5.21 (s, 2 H), 4.77 (s, 2 H),
1
3
.36 (s, 3 H); C NMR (75 MHz; DMSO; TMS) δ 153.04, 149.51, 147.43, 140.58, 140.24,133.19, 132.19,
30.45, 127.89, 122.15, 117.50, 116.57, 61.81, 56.11, 19.46; ESI-MS (m/z): calculated for C H ClN O [M
1
5
15
2
4
+
H] 322.75; found, 323;HRMS m/z: calculated for C H ClN O 322.75 found; 323.0820.
1
5
15
2
4
(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-yl)methyl(4-chlorophenyl)carbamate (4b)Light brownish
˚
solid (94%; 0.181 g); melting range = 154.0-156.0 C; R = 0.48 (hexane-EtOAc = 4/6 v/v); FT-IR (ATR)
f
-1
1
νcm : 3616, 3318, 2950, 2820, 1705, 1530, 1223, 737; H NMR (300 MHz; DMSO; TMS): δ 9.85 (s, 1 H),
1
3
7
.99 (s, 1 H), 7.41 - 7.56 (m, 2 H), 7.30 - 7.37 (m, 2 H), 5.20 (s, 2 H), 4.77 (s, 2 H), 2.37 (s, 3 H); C NMR
(75 MHz; DMSO; TMS) δ 153.08, 149.49, 147.39, 140.23, 138.01,132.13, 128.64, 127.94, 126.09, 119.63,
+
6
1.70, 56.13, 19.45; ESI-MS (m/z): calculated for C H ClN O [M + H] 322.75; found, 323;HRMS m/z:
1
5
15
2
4
calculated for C H ClN O 322.75 found; 323.0820.
1
5
15
2
4
(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-yl)methyl(4-bromophenyl)carbamate (4c)Brown solid
˚
-1
(
96%; 0.2 g); melting range = 169.0-171.0 C; R = 0.52 (hexane-EtOAc = 4/6 v/v); FT-IR (ATR) νcm :
f
1
3
1
1
616, 3349, 2951, 2820, 1710, 1537, 1219, 620; H NMR (300 MHz; DMSO; TMS): δ 9.86 (s, 1 H), 7.98 (s,
1
3
H), 7.39 - 7.50 (m, 5 H), 5.20 (s, 2 H), 4.77 (s, 2 H), 2.36 (s, 3 H); C NMR (75 MHz; DMSO; TMS) δ
53.05, 149.48, 147.39, 140.23, 138.43,132.13, 131.54, 127.92, 120.04, 114.01 61.71, 56.12, 19.45; ESI-MS
+
(
m/z): calculated for C H BrN O [M + H] 367.20; found, 369;HRMS m/z: calculated for
15
15
2
4
C H BrN O 367.20 found; 369.0281.
15
15
2
4
(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-yl)methyl(4-nitrophenyl)carbamate(4d)
Yellow
solid
˚
-1
(
92%; 0.180 g); melting range = 175.0-179.0 C; R = 0.47 (hexane-EtOAc = 4/6 v/v); FT-IR (ATR) νcm :
f
1
3
625, 3338, 2926, 2810, 1726, 1550, 1524, 1218; H NMR (300 MHz; DMSO; TMS): δ 10.73 (s, 1 H), 8.32
(s, 1 H), 8.21 - 8.24 (m, 1 H), 8.21 (m, 1 H), 7.72 (d, J=9.08 Hz, 2 H), 5.43 (s, 2 H), 4.87 (s, 2 H), 2.62 (s, 3
1
3
H); C NMR (75 MHz; DMSO; TMS) δ 153.05, 151.92, 145.34, 143.16, 141.84,141.74, 132.58, 130.84,
+
1
3
25.07, 117.78, 61.11, 55.56, 15.12; ESI-MS (m/z): calculated for C H N O [M + H] 333.30; found,
1
5
15
3
6
34.2;HRMS m/z: calculated for C H N O 333.30 found; 334.1059.
1
5
15
3
4
(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-yl)methyl(4-methoxyphenyl)carbamate (4e) Brown solid
˚
-1
(
91%; 0.150 g); melting range = 148.0-150.0 C; R = 0.55 (hexane-EtOAc = 4/6 v/v); FT-IR (ATR) νcm :
f
1
3
619, 3393, 2951, 2821, 1701, 1531, 1215; H NMR (300 MHz; DMSO; TMS): δ 9.49 (br s, 1 H), 7.98 (s, 1
H), 7.35 (br d, J=8.80 Hz, 2 H), 6.86 (d, J=9.08 Hz, 3 H), 5.17 (s, 2 H), 4.77 (s, 2 H),3.70 (s, 3 H), 2.36 (s, 3
1
3
H); C NMR (75 MHz; DMSO; TMS) δ 154.81, 153.29, 149.52, 147.26, 140.11, 132.02, 128.18, 119.80,

+
1
3
13.95, 61.38, 56.19, 55.13, 19.43; ESI-MS (m/z): calculated for C H N O [M + H] 318.33; found,
16 18 2 4
19.2;HRMS m/z: calculated for C H N O 318.33; found 319.1309.
1
6
15
2
4
(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-yl)methyl(3-chloro-4 methylphenyl)carbamate(4f). Light
˚
brownish solid (94%; 0.205 g); melting range = 167.0-170.0 C; R = 0.45 (hexane-EtOAc = 4/6 v/v); FT-IR
f
-1
1
(
ATR) νcm : 3625, 3391, 2954, 2821, 1709, 1543, 1219, 687; H NMR (300 MHz; DMSO; TMS): δ 9.81 (
s, 1 H), 7.98 (s, 1 H), 7.51 - 7.66 (m, 1 H), 7.19 - 7.32 (m, 2 H), 5.19 (s, 2 H), 4.77 (s, 2 H),2.36 (s, 3 H),
1
3
2
1
.25 (s, 3 H); C NMR (75 MHz; DMSO; TMS) δ 153.05, 149.49, 147.38, 140.24, 138.18, 133.08, 132.14,
31.20, 128.95, 127.94, 118.01, 116.83, 61.70, 56.11, 19.45, 18.76; ESI-MS (m/z): calculated for
+
C H ClN O [M + H] 336.77; found, 337.2;HRMS m/z: calculated forC H ClN O 336.77 ; found
16
17
2
4
16 17
2
4
3
37.0959.
(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-yl)methyl(4-chloro-
˚
3
0
(trifluoromethyl)phenyl)carbamate(4g). White solid (91%; 0.220 g); melting range = 164.0-166.0 C; R =
f
-1
1
.52 (hexane-EtOAc = 4/6 v/v); FT-IR (ATR) νcm : 3617, 3348, 2882, 2812, 2361, 1537,1118, 755; H
NMR (300 MHz; DMSO; TMS): δ 10.19 ( s, 1 H),7.95 - 8.06 (m, 2 H), 7.59 - 7.75 (m, 2 H), 5.23 (s, 2 H),
1
3
4
1
.77 (s, 2 H),2.37 (s, 3 H); C NMR (75 MHz; DMSO; TMS) δ 153.08, 149.48, 147.59, 140.25, 138.64,
32.25, 132.14, 127.76, 126.99, 123.11, 122.85, 116.66, 62.04, 56.05, 55.13, 19.46; ESI-MS (m/z): calcd for
+
C H ClF N O [M + H] 390.74; found, 391.2;HRMS m/z:calcd for C H ClF N O 390.74; found
16
14
3
2
4
16 14
3
2
4
3
91.0681.
(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-yl)methylallylcarbamate (4h). Light brownish solid (92%;
˚
-1
0
.120 g); melting range = 135.0-138.0 C; R = 0.46 (hexane-EtOAc = 4/6 v/v); FT-IR (ATR) νcm : 3619,
f
1
3
292, 2977, 2820, 1688, 1600, 1542, 1247; H NMR (300 MHz; DMSO; TMS): δ 7.88 ( s, 1 H), 7.40 (br t,
J=5.36 Hz, 1 H), 5.78 (ddt, J=17.19, 10.32, 5.09, 5.09 Hz, 1 H),4.99 - 5.15 (m, 4 H),4.70 (s, 2 H), 3.53 - 3.70
+
(
m, 2 H),2.34 (s, 3 H); ESI-MS (m/z): calculated for C H N O [M + H] 252.27; found, 253.2;HRMS
1
2
16
2
4
m/z:calcd forC H N O 252.27; found 253.1201.
1
2
16
2
4
(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-yl)methyl (3-phenylpropyl)carbamate(4i). Light brownish
˚
solid (94%; 0.150 g); melting range = 142.0-145.0 C; R = 0.55 (hexane-EtOAc = 4/6 v/v); FT-IR (ATR)
f
-1
1
νcm : 3628, 3309, 2929, 2865, 1681, 1542, 1258; H NMR (300 MHz; DMSO; TMS): δ 7.91 ( s, 1 H),7.12
-
7.32 (m, 6 H), 5.05 (s, 2 H), 4.72 (s, 2 H), 2.92 - 3.06 (t, 2 H), 2.35 (s, 3 H), 1.61 - 1.74 (m, 2 H); ESI-MS
+
(
m/z): calculated for C H N O [M + H] 330.38; found, 331.2; HRMS m/z:calcd forC H N O 330.38;
1
8
22
2
4
18 22
2
4
found 331.1679.
(5-hydroxy-4-(hydroxymethyl)-6-methylpyridin-3-yl)methyl(4-phenylbutyl)carbamate(4j) Brown solid (93%;
˚
-1
0
3
.130 g); melting range = 157.0-162.0 C; R = 0.49 (hexane-EtOAc = 4/6 v/v); FT-IR (ATR) νcm : 3606,
f
1
324, 2924, 2856, 1688, 1555, 1248; H NMR (300 MHz; DMSO; TMS): δ 7.99 ( s, 1 H),7.12 - 7.29 (m, 6
H), 5.03 (s, 2 H), 4.71 (s, 2 H), 2.95 - 2.59 (t, 2 H), 2.35 (s, 3 H), 1.49 - 1.57 (m, 2 H), 1.21 - 1.31 (m, 2 H);

+
ESI-MS (m/z): calculated for C H N O [M + H] 344.41; found, 345.2;HRMS m/z:calcd forC H N O
4
1
9
24
2
4
19 24
2
3
3
3
44.41; found 345.1834.
.2 In-vitro metal chelation studies
.2.1 Metal Chelation using UV-Visible Spectrophotometer
The UV spectra of all the compounds for metal chelation assay were carried out using dual beam UV-visible
spectrophotometer Agilent Technologies 500. All the salts (CuCl , ZnSO , Fe (SO4) ) were purchased from
2
4
2
3
Sigma Aldrich and Alfa Aesar. Absorbance was measured along the wavelength 200-600nm of compounds
alone or with metals were measured by keeping the concentration of the compounds 60µM, in 1cm quartz
cuvette. The concentration for individual bio-metals was also kept fixed and same of that of the compound.
The final reaction mixture resulted of both compound and bio-metal in the ratio 1:1.(25)After an incubation
time of 10mins of the reaction mixture, the absorbance was taken. The molar ratio method was followed to
take the absorbance reading at 245nm. The final concentration of 4g was kept fixed (60µM) and titration was
2
+
carried out with increasing concentration of Cu (1.5-90µM).
3
.2.2 Fluorescence and it’s effect on Fluorescent intensity due to metal chelation
Fluorescent studies were conducted on Fluorescence spectrophotometer Agilent Technologies 500 in 1cm
glass cuvette.Fluorescent intensity was recorded at an excitation wavelength 275nmand emission
wavelength 295-530nm at 520-620volts, keeping both excitation and emission slits at 10 for all the
derivatives. Compound concentration or metal salt concentration was kept same as 4.2.1 and same method
2
+
2+
3+
was followed for metal chelation assay of compounds with Zn , Cu , Fe . Incubation time for all the
reaction mixtures was kept 10mins and the readings were taken.
3
.2.3 Complexformation of 4g-Cu(II)
An appropriate amount of copper(II) chloride was added to the ethanolic solution of ligand 4g. The mixture
was refluxed for about 1hour. The precipate formed was filtered and washed with warm water. The complex
formed was further spectroscopicly analysed [32].
3
.3 In-silico ADME Prediction
All the pharmacokinetic parameters were estimated using the software ChemBioDraw Ultra14.0. The
number of rotatable bonds for derivatives 4a-j was calculated using moleinspirationcheminformatics. The
pharmacokinetics are very crucial for crossing the BBB and should obey Lipinski’s Rule of 5.
3
.4 Pharmacodynamic Prediction
For the pharmacodynamic
http://www.pharmaexpert.ru/passonline/). This software estimates the predicted activity spectrum of a
prediction,
PASS
online
software
was
used
(
compound as probable activity(Pa) and probable inactivity(Pi). Being probabilities, the Pa and Pi values vary
from 0.000 to 1.000 and, in general, Pa+Pi≠1,as these probabilities were calculated independently. The
PASS prediction results were interpreted and used in a flexible manner: (i) only activities with Pa > Pi are

considered as possible for a particular compound,(ii) if Pa > 0.7, the chance to find the activity is
experimentally high, (iii) if 0.5<Pa<0.7, the chance to find the activity experimentally is less, but the
compound is probably not so similar to known pharmaceutical agents; (iv) if Pa < 0.5, the chance to find the
activity is experimentally less, but the chance to find a structurally new compound , i.e, new chemical entity
is more [Chaudhary et al.; 2012; Mishra et al. 2015].
3
.5 Experimental determination of log P value of 4g
To determine the log P value, only 4g was taken and a calibration curve was constructed. A stock solution of
0mM was made using 1-octanol (purchased from Merck). Different dilutions were made within the
1
concentration range of 20-120µM. A known amount of compound was dissolved in octanol –water system
and partitioned using a separating flask. Both the aqueous layer and the organic layer were separated. From
the calibration curve, the straight line equation obtained was used to calculate the concentration in octanol
layer. And the weight 4g taken was substracted and the concentration of the compound in water layer was
calculated. Finally Log P value was determined using the formula Log P=concentration in octanol
layer/concentration in water layer.
3
.6 Fluorescence Quenching Study of BSA with 4g
BSA was purchased from HiMedia. A 0.1M phosphate buffer solution was made in triple milli Q water
keeping the pH 7.4. A primary stock solution of BSA (1mM) was prepared and from it 5µM was prepared as
the secondary stock solution. The primary stock solution of the probe was prepared as 10µM and from it the
reaction mixtures were prepared. The final reaction mixtures comprised 2.5µM of protein solution and the
probe solution (0.25-2.5µM) in the ratio 1:1. Fluorescence quenching studies were performedusing
Fluorescence Spectrophotometer Agilent Technologies 500, keeping the temperature at 37 degree Celsius,
the excitation and emission slits 5 and the volts as 600. The reaction mixtures were incubated for 30 seconds
and the readings were taken.
3
.7 Molecular Docking Study
Molecular Docking study was performed using AutoDock Tools (version 1.5.6) which helped us to find out
the binding site and the mode of binding of 4g with BSA. The RCSB Protein Data Bank (PDB ID:4f5s) was
used as a source of X-ray crystal structure of BSA. The probe structure of 4g was drawn using Chem3D
Ultra 14.0. Gasteiger partial charges were added to the probe. A grid spacing of 0.403Å and grid maps of
1
26 X 126 X 126 Å were generated using AutoDock Tools (version 1.5.6). All the AutoDock parameters
were kept to their default values. Genetic algorithm was used for docking calculations, keeping the number
of GA runs and energy evaluations 100 and 250000. The population size and the crossover rate were kept to
their default values. The dock model was optimized with the minimum energy. This optimized dock model
was viewed in PyMOL software.(21)

3
.8Acetylcholinesterase(AChE) binding study with Compounds
The AChE binding study was performed using dual-beam UV-visible spectrophotometer Agilent
Technologies 500 in 1cm quartz cuvette. From a stock solution of 10mM of ligand, 60µM concentration
solution was made taking phosphate buffer solution (pH 7.4) as a medium for dilution. AChE solution was
added so that 1:1 ligand to enzyme ratio is maintained. After an incubation time of 30 seconds, spectrum was
recorded.
4
. Conclusion
In summary, we have designed and synthesized pyridoxine-carbamates4(a-j) by reacting protected
pyridoxine with isocyanates followed by deprotection of the acetonide ring, resulting in a series of
compounds. Biophysical experiments for metal-chelation using UV-Visible Spectrophotometer proved the
designed compound to be a better metal chelator when pyridoxine alone is considered. The carbamate
moiety along with the free –OH present in the compounds contributed towards metal chelation as interpreted
from the IR spectra. The effects of substitution on absorbance and fluorescence spectra were keenly studied
and explained. We even observed shifts in emission spectra with few compounds which is contemplated to
be a reason to act as a donor for the metal ion.4g was found the best one. Hereby, we report a new compound
4
gwhich has the ability to chelate metals. Further findings revealed the 1:1 complex formation between
metal and ligand 4g. Biophysical experiments with BSA and our probe 4gexhibited quenching phenomenon.
Detailed analysis of data gave us the static quenching mechanism to play a role in quenching. Molecular
docking studies confirmed that the quenching mechanism was due to the conformational changes of the
protein because of binding of the probe and not due to the direct interaction of the probe with the tryptophan
residues. 4g also showed binding with acetylcholinesterase enzyme, validated by UV-Visible spectrum. The
synthesized series has properties of drug-likeness and all of them passed the Lipinski Rule of five.
Acknowledgments
Authors would like to thank Dr. SJS Flora for supporting this work, Pooja Patil and Tiyas Pal are thankful to
the Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India for
providing fellowship.The authors are also thankful to Central Instrumentation Facility of NIPER-R for
characterization and allowing to perform spectral studies. NIPER-R/ Communication/118.
5
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