Design, Synthesis and Molecular Docking of Vanillic Acid Derivatives as Amylolytic Enzyme Inhibitors

Article abstract of DOI:10.1007/s11094-021-02439-4

In the present work, a series of vanillic acid derivatives have been synthesized and tested to exhibit promising amylolytic enzymes inhibition. Structures of the synthesized derivatives were studied by FT-IR, ¹H NMR, ¹³C NMR, EI-MS,

Full text of DOI:10.1007/s11094-021-02439-4

DOI 10.1007/s11094-021-02439-4
Pharmaceutical Chemistry Journal, Vol. 55, No. 5, August, 2021 (Russian Original Vol. 55, No. 5, May, 2021)
DESIGN, SYNTHESIS AND MOLECULAR DOCKING
OF VANILLIC ACID DERIVATIVES
AS AMYLOLYTIC ENZYME INHIBITORS
Nishi Gupta,1,2,* Alok Mukerjee,1 and Shanti Bhushan Mishra1
Original article submitted November 11, 2020.
In the present work, a series of vanillic acid derivatives have been synthesized and tested to exhibit promising
amylolytic enzymes inhibition. Structures of the synthesized derivatives were studied by FT-IR, 1H NMR, 13
C
NMR, EI-MS, and structural analysis. In order to confirm the interaction between ligand and receptor mole-
cule, in silico docking was performed. Results indicated that two derivatives (3a and 3g) are promising com-
pounds exhibiting good glide score to the development of selective inhibition of a-amylase and a-gluco-
sidase.
Keywords: alpha-amylase, alpha-glucosidase, in-silico studies, vanillic acid derivatives.
1. INTRODUCTION
antidiabetic effects [3]. Some natural products obtained from
plants can be used as small-molecule drug precursors, which
Diabetes mellitus is a metabolic disorder characterized
by hyperglycemia, glycosuria and negative nitrogen balance
and it is mainly due to absolute deficiency or diminished ef-
fectiveness of insulin. Diabetes is still not completely curable
by the present antidiabetic therapy. Insulin therapy is the
only satisfactory approach to diabetes mellitus, even though
it has several drawbacks like insulin resistance, anorexia,
brain atrophy, and fatty liver in chronic treatment [1]. Several
drugs such as sulfonylureas and biguanides are presently
available to reduce hyperglycemia in diabetes mellitus. Since
these drugs demonstrated significant side effects, the search
for a new class of compounds is essential to overcome these
problems. Therefore, the urgent need to look for new drug
scaffolds with minimum side effects is still a challenge to the
medicinal chemists [2].
can be converted into compounds of interest by chemical
modification or fermentation methods. The semisynthetic ap-
proach is usually employed to resolve the shortage of supply
due to the low yield of compounds from plants and/or the
high cost of total synthesis [4].
Phenolic acids are hydroxylated derivatives of benzoic
and cinnamic acids. Vanillic acid (4-hydroxy-3-methoxy-
benzoic acid, Fig. 1) is a dietary phenolic compound found in
plants and fruits and an intermediate in the production of
vanillin from ferulic acid. Vanillic acid has been previously
reported in scientific investigations to exhibit beneficial ef-
fects such as antioxidant, antimutagenic, anti-cancer, anti-in-
flammatory, and neuroprotective [5, 6]. Moreover, these
phytochemicals significantly reduced expression of the en-
zymes involved in fatty acid and cholesterol synthesis, indi-
cating that they might help prevent the secondary complica-
Nowadays, many medicinal plants have been reported to
be useful in treating diabetes. Antihyperglycemic activity of
natural products is mainly due to their ability to restore the
function of pancreatic tissues by causing increase in the insu-
lin output, decrease the absorption of glucose in intestine, or
facilitate metabolites in insulin dependent processes. Most of
the useful plants contain glycosides, alkaloids, terpenoids,
flavonoids, carotenoids, etc., which frequently exhibit
1
United Institute of Pharmacy, Prayagraj, Allahabad, Uttar Pradesh,
211010 India.
2
Dr. APJ Abdul Kalam Technical University, Lucknow, Uttar Pradesh,
226031 India.
*
e-mail: nishi0032@gmail.com
Fig. 1. Chemical structure of vanillic acid.
427
0091-150X/21/5505-0427 © 2021 Springer Science+Business Media, LLC

428
Nishi Gupta et al.
Fig. 2. Synthesis of vanillic acid derivatives.
tions of diabetes [7]. Since a new diabetes management strat-
egy is needed that is more effective and has fewer side
effects, we made an attempt to synthesize various derivatives
of vanillic acid as potent oral hypoglycemic agents.
DMSO-d as deuterated solvents using tetramethylsilane
6
(TMS) as the internal standard. Chemical shift values are
given in d (ppm) scale and the signals are described as s (sin-
glet), d (doublet), t (triplet), q (quartet) and m (multiplet)
whereas coupling constants (J) are expressed in Hz. Mass
spectra (ESI-MS) were recorded on Waters mass spectrome-
ter (Q-TOF Premier).
2. EXPERIMENTAL CHEMICAL PART
2.1. Chemicals and Instruments
2.2. General Procedure for Synthesis of Compounds 3a-3k
All the chemicals, solvents and reagents used were pur-
chased from Merck, India. Progress of the reaction was mon-
itored by TLC performed on silica gel G and the spots were
exposed to iodine vapor and UV lamp for visualization using
Chloroform: Ethyl acetate: formic acid (5:4:1) as mobile
phase. Fourier Transform Infra-Red spectra were recorded on
(Perkin Elmer Spectrum version 10.03.02).Proton (1H)
NMR spectra were recorded on JEOL model on 400 MHz.
Chlorination of vanillic acid (1) has been carried out by
refluxing with thionyl chloride to 4-hydroxy-3-methoxy-
benzoyl chloride (2). Chloride (2) was further treated with
substituted amines in presence of base to produce corre-
sponding amides. These reactions led to the formation of
compounds 3a-3k (Fig. 2). The separated solids were filtered
under reduced pressure and washed thoroughly with water
[8].
All the spectra were recorded at 25°C using CDCl and
3
Fig. 3. Structure of 4-hydroxy-3-methoxybenzoylchloride (2).
Fig. 4. Structure of substituted amides.

Design, Synthesis and Molecular Docking
429
Synthesis of 4-hydroxy-3-methoxybenzoyl chloride
(2). 4-Hydroxy-3-methoxybenzoic acid 1 (1.5 gm), 25 mL
thionyl chloride, and a drop of dimethyl formamide (DMF)
were refluxed in a boiling water bath for 2 h to yield chloride
2 (Fig. 3).
carbon tetrachloride, methanol https://www.rcsb.org/struc-
ture/3IJ8.; insoluble in acetone, butanol; M. P.: 282 – 285°C;
IR spectrum (nmax
,
cm–1): 1550 – 1510 (CO-NH),
1600 – 1700 (-CO stretch), 3500 (bonded –OH stretch alco-
hol), 1200 – 1275 (ether –CO stretch). MS (ESI), m/z:
244.194, 169.059, 150.225, 134.969, 189.041; 1H NMR
spectrum in DMSO (d, ppm): 7.38 (t, Ar-H),7.34 – 7.50 (m,
5H, Ar-H), 3.79 (s, 3H), 2.48 – 2.51 (s, Ar-C-H).
Synthesis of vanillic acid analogs 3a-3k. 4-hydroxy-3-
methoxybenzoyl chloride 2 (0.055 mol) was added in small
portion to a solution of amine (0.11 mol) and 60 mL of 10%
potassium hydroxide with constant stirring and intermittent
cooling. The separated solid was then filtered at the vacuum
pump and washed thoroughly with water. The crude product
was filtered, washed with water and then recrystallized using
chloroform.
N-(3-Chlorophenyl)-4-hydroxy-3-methoxybenzamide
(3e): Molecular formula-:C H O Cl; Yield: 2.09 g (85%);
14 12
3
Color: brown amorphous powder; Solubility: soluble in ace-
tone, water; insoluble in methanol, butanol, chloroform, car-
bon tetrachloride; M. P.: 295 – 298°C; IR spectrum (nmax, cm
–1): 1550 – 1510 (CO-NH), 1550 (N-O stretch), 1600 – 1700
(-CO stretch), 3500 (bonded –OH stretch alcohol),
1200 – 1275 (ether –CO stretch), 680 (m-substituted ben-
zene). MS (ESI), m/z: 278.150, 169.127, 131.12, 127.813. 1H
2.3. Spectral Data of Compounds 3a-3k
4-hHdroxy-3-methoxybenzohydrazide(3a). Molecular
formula: C H O N ; % Yield: 1.37 g (85%); Color: dark
8
10
3
2
yellowish powder; Solubility: soluble in acetone, methanol
and butanol; insoluble in water, chloroform, carbon tetra-
chloride; M. P.: 291 – 295°C; IR spectrum (nmax, cm–1):
1550 – 1510 (CO-NH), 1600 – 1700 (-CO stretch), 3500
(bonded –OH stretch alcohol), 1200 – 1275 (ether –CO
stretch), 1600 (aromatic C-C ring stretch). MS (ESI), m/z:
183.236, 151.035, 154, 128.015; 1H NMR spectrum in
DMSO (d, ppm): 6.83 – 7.66 (Ar-H), 3.93 (d, H), 3.80 (s,
3H), 2.5 (m), 2.49 – 2.53 (s, Ar-C-H)
NMR spectrum in CDCl (d, ppm): 7.05 – 7.1 (m, Ar-H,
3
5H), 6.54 – 6.59 (M, 5H), 3.87 – 3.88 (d, H), 3.67 (s, H).
4-Hydroxy-3-methoxy-N-(3-nitrophenyl)benzamide
(3f). Molecular formula; C H O N ; Yield: 2.03 g (79%);
14 12
5
2
Color: dark yellowish powder; Solubility: soluble in acetone,
methanol and butanol; insoluble in water, chloroform, carbon
tetrachloride; M. P.: 278 – 279°C; IR spectrum (nmax, cm–1):
1550 – 1510 (CO-NH), 1550 (N-O stretch), 1600 – 1700
(-CO stretch), 3500 (bonded –OH stretch alcohol),
1200 – 1275 (ether –CO stretch), 680 (m-substituted ben-
zene). MS (ESI), m/z: 282.336, 262.691, 230.086, 207.201,
4-Hydroxy-3-methoxy-N’-phenylbenzohydrazide (3b).
Molecular formula: C H O N ; Yield: 2.07 g (90%);
14 14
3
2
121.062, 169.262, 139.086. 1H NMR spectrum in CDCl (d,
Color: golden yellow crystals; Solubility: soluble in acetone,
methanol and butanol; insoluble in water, chloroform, carbon
tetrachloride; M. P.: 282 – 290°C; IR spectrum (nmax, cm–1):
1550 – 1510 (CO-NH), 1600 – 1700 (-CO stretch), 3500
(bonded –OH stretch alcohol), 1200 – 1275 (ether –CO
stretch), 1600 (ArC-C ring stretch).MS (ESI), m/z: 259.248,
3
ppm): 8.13 (dd, Ar-H), 8.1 – 8.13 (dd, o/m), 7.72 (d, Ar-H),
7.69 (d, Ar-H), 7.58 – 7.59 (dd, o/m), 7.33 – 7.38 (dt, di
o/m), 7.38 (t, Ar-H), 7.261 (CDCl ), 6.98 (s), 6.95 (s),
3
6.68 – 6.79 (dt, Ar-H), 6.81 – 6.82 (dd, Ar-H), 6.04 (N-H),
3.96 (s, OCH ), 1.19 – 1.33 (monosubstituted benzene ring).
3
1
133.146, 149.145, 163.186, 177.093; H NMR spectrum in
N-(4-Bromophenyl)-4-hydroxy-3-methoxybenzamide
CDCl (d, ppm): 7.72 (d, H), 7.69 (d, H), 7.58 – 7.59 (dd,
(3g). Molecular formula: C H O NBr; Yield: 2.48 g
3
14 12
3
o/m), 7.261 (s, CDCl ), 6.95 – 7.72 (Ar-H), 6.95 (s, H), 6.98
(87%); Color: green yellowish powder; Solubility: soluble in
acetone, methanol, butanol. Insoluble in water, chloroform
and carbon tetrachloride; M. P.: 284 – 285°C; IR spectrum
(nmax, cm–1): 1550 – 1510 (-CO-NH), 1550 (N-O stretch),
1600 – 1700 (-CO stretch), 3500 (bonded –OH stretch alco-
hol), 1200 – 1275 (ether –CO stretch), 650 – 800
(p-disubstituted), 1600(aromatic C-C ring stretch). MS
(ESI), m/z: 322.097,229.140, 215.099, 128.015, 169.127,
3
(s, H), 3.95 (d, H), 3.80 (s, 3H), 1.25 (monosubstituted ben-
zene ring).
4-Hydroxy-3-methoxy-N¢-(2,4-dinitrophenyl)benzo-
hydrazide (3c). Molecular formula: C H O N ; Yield:
14 11
7
3
2.61 g (88%); Color: brown amorphous powder; Solubility:
soluble in water, acetone; insoluble in butanol, methanol,
chloroform, carbon tetrachloride; M. P.: 300 – 302°C; IR
spectrum (nmax, cm–1): 1550 – 1510 (CO-NH), 1550 (N-O
stretch), 1600 – 1700 (-CO stretch), 3500 (bonded –OH
stretch alcohol), 1200 – 1275 (ether –CO stretch), 1600 (aro-
matic C-C ring stretch). MS (ESI), m/z: 329.118, 222.120,
1
175.068 &173.920; H NMR spectrum in CDCl (d, ppm):
3
7.72 (dd, H),, 6.66 – 7.72 (Ar-H), 7.69 (d, H), 7.58 – 7.59
(dd, o/m), 7.33 – 7.34 (dt, di o/m), 7.26 (CDCl ), 6.98 (s),
3
6.95 (s), 6.79 (dd, Ar-H), 3.96 (s, OCH ), 2.12 – 2.74
3
1
204.973, 282.336, 154.815, 125.113, 245.005, 181.076; H
(Ar-C-H), 1.25 – 1.33 (monosubstituted benzene ring).
4-Hydroxy-3-methoxy-N-(2-nitrophenyl)benzamide
(3h). Molecular formula: C H O N ; Yield: 2.13 g (83%);
NMR spectrum in CDCl (d, ppm): 7.261 (s, CDCl ), 3.93 (d,
3
3
H), 3.49 (s, OCH ), 1.56 (s, R CH), 1.25 – 1.33 (s, monosub-
14 12
5
2
3
3
Color: orange amorphous powder; Solubility: soluble in wa-
ter, acetone. insoluble in chloroform, carbon tetrachloride
methanol, butanol; M. P.: 279 – 281°C; IR spectrum (nmax
cm–1): 1550 – 1510 (-CO-NH), 1550 (N-O stretch),
stituted benzene ring), 0.82 – 0.88 (shielded H).
4-Hydroxy-3-methoxy-N-phenylbenzamide (3d). Molec-
ular formula: C H O N; Yield: 1.96 g (91%); Color: brown
,
14 13
3
amorphous powder; Solubility: soluble in water, chloroform,

430
Nishi Gupta et al.
1600 – 1700 (-CO stretch), 3500 (bonded –OH stretch alco-
hol), 1200 – 1275 (ether –CO stretch), 650 – 800
(p-disubstituted), 1600 (aromatic C-C ring stretch). MS
(ESI), m/z: 282.336, 262.691, 230.086, 207.201, 121.062,
3. EXPERIMENTAL PHARMACOLOGICAL PART
3.1. In Vitro Antidiabetic Activity
Alpha-amylase inhibitory activity of synthesized de-
rivatives (3f-3k). The a-amylase inhibitory activity of syn-
thesized derivatives was performed by Sigma – Aldrich kit
with slight modification [9]. The inhibitory activity of differ-
ent concentrations of synthesized derivative (3a) (10 –
100 µg/ml) was determined against a-amylase. The enzyme
(0.5 %) was prepared in phosphate buffer (pH 6.8). Briefly,
500 mL of different concentrations of synthesized derivatives
and 500 mL of 0.1M phosphate buffer (pH 6.8) containing
a-amylase were incubated at 25°C for 10 min. After pre-in-
cubation, 500 mL of 1% starch solution in 0.1 M phosphate
buffer (pH 6.8) was added to each tube and further incubated
at 25°C for 10 min. The reaction was stopped by addition of
1 mL of dinitrosalicylic acid reagent. The same was per-
formed for control where drug solution was replaced with
buffer. The test tubes were placed in a boiling water bath for
10 minutes and cooled. To each tube, 10 mL of distilled wa-
ter was added and the absorbance was measured spectropho-
tometrically at 540 nm. The percentage (%) inhibition of en-
zyme activity was calculated using the following formula:
1
169.262, 139.086, 180.131. H NMR spectrum in CDCl (d,
3
ppm): 8.08 – 8.11 (dd, o/m), 7.34 – 7.37 (dt, di o/m), 7.26
(CDCl ), 6.8 – 6.83 (dd, Ar-H), 6.68 – 6.79 (dt, Ar-H), 3.94
3
(s, OCH ).
3
N-(2-Chlorophenyl)-4-hydroxy-3-methoxybenzamide
(3i). Molecular formula: C H O NCl; Yield: 2.02 g (82%);
14 12
3
Color: green yellowish powder; Solubility: soluble in water,
chloroform, carbon tetrachloride, methanol; insoluble in ace-
tone, butanol; M. P.: 302 – 304°C; IR spectrum (nmax, cm–1):
1550 – 1510 (-CO-NH), 1550 (N-O stretch), 1600 – 1700
(-CO stretch), 3500 (bonded –OH stretch alcohol),
1200 – 1275 (ether –CO stretch), 735 – 770 (o-disub-
stituted), 1600 (aromatic C-C ring stretch). MS (ESI), m/z:
1
278.150, 169.127, 131.121, 127.813,183.01. H NMR spec-
trum in CDCl (d, ppm): 7.33 – 7.38 (dt, di o/m), 7.26
3
(CDCl ), 6.68 – 6.79 (dt, Ar-H), 6.81 – 6.82 (dd, Ar-H),3.95
3
(s, OCH ), 2.12 – 2.74 (Ar-C-H), 1.17 – 1.77 (monosubsti-
3
tuted benzene ring), 0.79 – 0.9 (shielded H).
N-(2-Aminophenyl)-4-hydroxy-3-methoxybenzamide
(3j). Molecular formula: C H O N ; Yield: 1.79 g (78%);
Inhibition of a-amylase activity (%) =
14 14
3
2
= [A Control – A Drug/A Control] ´ 100%.
Color: black amorphous powder; Solubility: soluble in water,
acetone; insoluble in chloroform, carbon tetrachloride meth-
anol, butanol; M. P.: 293 – 295°C; IR spectrum (nmax, cm–1):
1500 – 1510 (-CO-NH), 1550 (N-O stretch), 1600 – 1700
(-CO stretch), 3500 (bonded –OH stretch alcohol),
1200 – 1275 (ether –CO stretch),1600 (aromatic C-C ring
stretch), 800 – 1000 (N-H wagging), 1620 (N-H bending),
1175 (C-N aromatic stretch). MS (ESI), m/z: 241.157,
540
540
540
The IC value was determined from a plot of the per-
50
centage inhibition versus sample concentration. Acarbose
was used as the standard control and the experiment was per-
formed in duplicate.
Alpha-glucosidase Inhibitory Activity of synthesized
derivatives (3a-3k). The a-glucosidase inhibition assay was
performed according to the standard procedure with slight
modification [10]. a-Glucosidase (2 U/mL) was premixed
with 20 mL of test sample at various concentration and incu-
bated for 5 min at 37°C. 1 Mm para-nitrophenylglucopyra-
noside (20 mL in 50mM of phosphate buffer (pH 6.8) was
added to initiate the reaction. The mixture was further incu-
bated at 37°C for 20 min. The reaction was terminated by ad-
dition of 50 mL of 1 M sodium carbonate and the final vol-
ume was made up to150 mL. The a-glucosidase activity was
determined spectrophotometrically at 405 nm by measuring
the amount of para-nitrophenol released from pNPG. The as-
say was performed in triplicate. The concentration of test
sample required to inhibit 50% of alpha glucosidase activity
1
211.116, 154.951, 119.104 H NMR spectrum in DMSO (d,
ppm): 8.0 (dd, o/m), 7.7 (dd, 2H), 7.63 – 7.68 (d, H), 3.9 (s,
OCH ), 2.4 – 2.5 (Ar-H).
3
4-Hydroxy-3-methoxy-N-(3-methoxyphenyl)benzami
de (3k). Molecular formula: C H O N; Yield: 1.97 g
15 15
4
(81%); Color: black amorphous powder; Solubility: soluble
in water, acetone. insoluble in chloroform, carbon tetrachlo-
ride methanol, butanol; M. P.: 280 – 281°C; IR spectrum
(nmax, cm–1): 1500 – 1510 (-CO-NH), 1550 (N-O stretch),
1600 – 1700 (-CO stretch), 3500 (bonded –OH stretch alco-
hol), 1200 – 1275 (ether –CO stretch),1600 (aromatic C-C
ring stretch), 800 – 1000 (N-H wagging), 1620 (N-H bend-
ing), 1175((C-N aromatic stretch), 680 (m-substituted ben-
zene). MS (ESI), m/z: 274.167, 253.038, 244.059, 207.876,
179.118, 172.165, 154.951, 130.108, 128.015. 1H NMR
under the conditions was defined as the IC value. The ex-
50
periment was repeated thrice with same protocol. The results
are presented in Table 1.
3.2. Molecular Docking Studies
spectrum in CDCl (d, ppm): 7.72 (d, H), 7.69 (d, H), 7.58
3
(dd, o/m), 7.38(t, H), 7.31 (dt),7.26 (CDCl ),6.95 (s),
In this study, all the designed compounds were subjected
to molecular docking to as determine their binding mode at
human pancreatic alpha amylase. The biological target mole-
3
6.87 – 6.88 (dd, Ar-H), 3.97 (s, OCH ), 1.25 – 1.28 (mono-
3
substituted benzene), 0.82 – 0.85 (shielded H).

Design, Synthesis and Molecular Docking
431
TABLE 1. Inhibitory Effects of Vanillic Acid Derivatives against a-Amylase and a- Glucosidase
IC50 mmol/mL
Binding energy
(kcal/mol)
Analog
Structure
IC50 mmol/mL a-amylase
3.73 ± 0.01
a- glucosidase
3a
–6.4
4.54 ± 0.021
3b
–4.02
5.81 ± 0.002
6.31 ± 0.0184
3c
–3.58
5.82 ± 0.002
6.21 ± 0.0028
3d
–5.36
5.56 ± 0.021
6.89 ± 0.0008
3e
–4.9
5.14 ± 0.001
5.52 ± 0.013
3f
–1.33
5.78 ± 0.019
5.99 ± 0.002
3g
–4.75
4.92 ± 0.012
5.35 ± 0.006
3h
–2.03
5.85 ± 0.028
5.51 ± 0.0001

432
Nishi Gupta et al.
IC50 mmol/mL
Binding energy
(kcal/mol)
Analog
Structure
IC50 mmol/mL a-amylase
6.60 ± 0.001
a- glucosidase
3i
–1.18
5.42 ± 0.002
3j
–4.02
5.74 ± 0.002
5.14 ± 0.011
3k
–5.44
5.07 ± 0.002
7.00 ± 0.003
Acarbose
7.36
6.9
–6.8
1
posed structures were confirmed by H NMR, melting point
cule was downloaded from the protein data bank, PDB ID:
3IJ8 (Fig. 5) at a resolution of 1.43Å [11].The target was pre-
pared for docking by removing all heteroatoms, nonreceptor
atoms, water and other ions, etc. Molecular docking was per-
formed for designed compounds 3a–3k as potential a-amy-
lase inhibitors. Compounds 3a–3k were simulated using
ChemDraw Ultra 8.0 software and converted into suitable
model structures. The energy minimized structures are re-
quired for molecular docking and for the preparation of cor-
responding PDB files. Docking studies were performed on
prepared ligands to predict the binding energy to find out the
most acceptable docked pose. Confirmation of docking was
done using Mcule platform.
(M.P.) values, and mass spectrometry data.
4.2. In Vitro Anti-Diabetic Assay of Vanillic Acid Analogs
In this experiment, a-amylase and a-glucosidase has
been selected to determine the anti-diabetic activity of all
tested derivatives of vanillic acid with a slight modification.
Acarbose was selected as positive control in the experiment.
The group of chief intestinal enzymes like a-amylase and
a-glucosidase were involved in the digestion of carbohy-
drates. Acarbose competitively inhibit these enzymes. Oral
hypoglycemic drugs act mainly by inhibiting saccharide-hy-
drolyzing enzymes (alpha-amylase and alpha-glucosidase) in
type-II diabetes. Digestion of carbohydrates can be pro-
longed through the inhibitions of these enzymes, resulting in
4. RESULTS AND DISCUSSION
4.1. Chemistry
Initially, Carboxylic group of vanillic acid react with
thionyl chloride to produce 4-hydroxy-3-methoxybenzoyl-
chloride (2) according to the literature procedure [8]. During
this reaction, the hydroxyl group of carboxylic acid is con-
verted to a chlorosulfite intermediate making it a better leav-
ing group. The chloride anion produced during the reaction
acts a nucleophile. 4-Hydroxy-3-methoxybenzoylchloride 2
was reacted with potassium hydroxide and substituted
amines to prepare the desired final derivatives 3a-3k de-
picted in Fig. 4. All compounds were purified and the pro-
Fig. 5. 3D structure of pancreatic a-amylase PDB ID: 31J8.

Design, Synthesis and Molecular Docking
433
Fig. 6. Binding mode of acarbose docked with a-amylase.
Fig. 7. Interaction and superimposed structure of 4-hydroxy-3-methoxybenzohydrazide (3a) with a-amylase amino acid residue.
Fig. 8. Interaction and superimposed structure of 4-hydroxy-3-methoxybenzohydrazide (3a) with a-amylase surface.
the reduction of glucose absorption rate and postprandial
plasma glucose level.
zymatic inhibitory activity, especially analog 3a with the
IC values of 3.71 and 4.56 mmol/mL and compound 3g
50
Different concentrations of vanillic acid derivatives were
subjected to alpha-amylase and alpha-glucosidase inhibitory
assay. The results are presented in Table 1. These data sug-
gested that most of the derivatives exhibited significant en-
with the IC values of 4.92 and 5.35 mmol/mL against al-
50
pha-amylase and alpha-glucosidase, respectively. These val-
ues are lower than those for other analogs and acarbose as
the positive control.

434
Nishi Gupta et al.
Fig. 9. Interaction and superimposed structure of N-(4-bromophenyl)-4-hydroxy-3-methoxybenzamide (3g) with a-amylase amino acid resi-
due.
As shown in Figs. 6, 7, 8, and 9, acarbose and synthe-
sized compounds 3a and 3g are involved in similar binding
interactions with the target protein molecule. Figure 6 shows
that acarbose can form hydrogen bonds with ALA198,
ASP197, TYR163, ILE235 and HIS305residues in the active
site. While 3a can interact with ALA198, ASP197, HIS305,
TYR62 and GLN63 (Fig. 7) residues, 3g interacts with
VAL198, ASP197, ALA198, GLU233, SER199 (Fig. 9). The
binding free energy of acarbose was –6.8 kcal/mol and those
for analogs 3a and 3g were –6.4 and –4.75 kcal/mol, respec-
tively [13].
The experimentally most active compounds also exhib-
Fig. 10. Correlation plots of IC50 and docking score of vanillic acid
ited a good correlation coefficient (r2 = 0.643) between the
derivatives vs. a-amylase.
docking score and IC values of the ligands (Fig. 10). There-
50
fore, analog 3a has significant inhibitory activity against
a-amylase, which is also confirmed by in vitro enzymatic
model.
4.3. Molecular Docking Studies
Thus it can be postulated that the compounds 3a and 3g
In order to study the binding interactions between alpha
can be explored for in vivo activity in the near future. These
amylase with the ligands, all the compounds were docked
studies will be helpful for further lead optimization and de-
and the binding interactions were calculated. To determine
signing of new amylolytic inhibitors for the treatment of dia-
the complete intermolecular interactions between the ligand
betes.
and target protein (31J8) (Fig. 5), an automated and virtual
screening program AutoDock was used. Docking is a combi-
ACKNOWLEDGMENTS
nation of algorithms and scoring functions. It performs
grid-based ligand docking with energetics and finds for fa-
Authors are grateful to Mrs. Shradhanjali Singh, Associ-
vorable interactions between one or more typically small
ate Professor, United Institute of Pharmacy for serving their
ligand molecules and a typically larger receptor molecule,
practical hands in spectroscopic studies and interpretation of
usually a protein. Three dimensional structural information
data.
of the target was taken from the Protein Data Bank (PDB).
Compound structures were built with Chem Draw Ultra 10.0
CONFLICT OF INTEREST
[12]. The receptor preparation was done to delete water mol-
ecules not related to active sites and also to regenerate the
The authors declare that they have no conflicts of inter-
native status, and also for the addition of hydrogen atoms.
est.

Design, Synthesis and Molecular Docking
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