Sulfonamide hybrid schiff bases of anthranilic acid: Synthesis, characterization and their biological potential

Article abstract of DOI:10.1016/j.molstruc.2019.02.056

In the present work, the novel Schiff bases (03–20) of 4-chloro-N-[2-(hydrazinocarbonyl) phenyl]benzenesulfonamide (02) were synthesized by reacting it with various aldehydes. 4-Chloro-N-[2-(hydrazinocarbonyl) phenyl]benzenesulfonamide (02) was synthesize

Full text of DOI:10.1016/j.molstruc.2019.02.056

Accepted Manuscript
Sulfonamide hybrid schiff bases of anthranilic acid: Synthesis, characterization and
their biological potential
Naghmana Kausar, Shahzad Muratza, Muhammad Asam Raza, Hummera Rafique,
Muhammad Nadeem Arshad, Ataf Ali Altaf, Abdullah M. Asiri, Syed Salman Shafqat,
Syed Rizwan Shafqat
PII:
S0022-2860(19)30187-5
DOI:
https://doi.org/10.1016/j.molstruc.2019.02.056
MOLSTR 26213
Reference:
To appear in: Journal of Molecular Structure
Received Date: 15 November 2018
Revised Date: 13 February 2019
Accepted Date: 14 February 2019
Please cite this article as: N. Kausar, S. Muratza, M.A. Raza, H. Rafique, M.N. Arshad, A.A. Altaf,
A.M. Asiri, S.S. Shafqat, S.R. Shafqat, Sulfonamide hybrid schiff bases of anthranilic acid: Synthesis,
characterization and their biological potential, Journal of Molecular Structure (2019), doi: https://
doi.org/10.1016/j.molstruc.2019.02.056.
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ACCEPTED MANUSCRIPT
Sulfonamide Hybrid Schiff Bases of Anthranilic acid: Synthesis,
Characterization and their Biological Potential
*
a
a
a
a
Naghmana Kausar , Shahzad Muratza , Muhammad Asam Raza , Hummera Rafique ,
*
Muhammad Nadeem Arshad b, c, Ataf Ali Altaf a, Abdullah M. Asiri b, c , Syed Salman Shafqat d,
Syed Rizwan Shafqat e
a Department of Chemistry, University of Gujrat, 50700, Gujrat, Pakistan.
b Chemistry Department, Faculty of Science, King Abdulaziz University, P. O. Box 80203,
Jeddah 21589, Saudi Arabia.
c Center of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University,
P. O. Box 80203, Jeddah 21589, Saudi Arabia
d Department of Chemistry, University of Management and Technology, Sialkot, Pakistan.
e University Malaysia Sarwak (Unimas), Malaysia.
.
Corresponding Author:
Dr. Shahzad Murtaza
Associate Professor, Department of Chemistry,
Faculty of Science, University of Gujrat, Gujrat, Pakistan
Email: shahzad.murtaza@uog.edu.pk

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Abstract:
In the present work, the novel Schiff bases (03-20) of 4-chloro-N-[2-(hydrazinocarbonyl)
phenyl]benzenesulfonamide (02) were synthesized by reacting it with various aldehydes. 4-
chloro-N-[2-(hydrazinocarbonyl) phenyl]benzenesulfonamide (02) was synthesized by reacting
methyl 2-{[(4-chlorophenyl)sulfonyl]amino}benzoate (01) with hydrazine. All synthesized
compounds (01-20) were characterized by using FTIR, NMR and Mass spectrometry and by
single crystal X-ray diffraction (XRD) analysis techniques. The synthesized compounds were
screened for their enzyme inhibition potential against AChE and BChE enzymes. Molecular
docking studies were carried out to demonstrate putative binding modes. Antioxidant potential of
the synthesized compounds was also determined. Enzyme inhibition assay revealed that
compounds 02 and 12 showed maximum inhibition against AChE enzyme with percentage
inhibition of 91% and 83% respectively, while compounds 12 and 07 showed highest inhibition
against BChE with percentage inhibition of 92% and 81% respectively. Molecular docking
studies supported the results of enzyme inhibition assay with binding energy values of -8.49 Kcal
mol-1 against AChE and -8.39 Kcal mol-1 against BChE for compound 12. Antioxidant studies
also showed good results with percentage scavenging of 96% for both compounds 02 and 19
investigated by DPPH scavenging method.
Keywords: sulfonamide, mental disorders, docking studies, hydrazine, methyl anthranilate,
DPPH
Graphical Abstract:

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1. Introduction
The revolutions in the field of research have explored many secret areas that have the potential to
resolve many known problems of the advanced world. Many achievements have been gained in
medical sciences, such as cure of many fetal diseases. The importance of developing new drugs,
synthesizing new materials having active pharmacophore is a better strategy. Sulfonamide and
Schiff base are the two well-known moieties present in active drugs (Fig. 1) [1].
NH2
O
S
O
NH2
H
N
N
F
O
S
H
N
O
O
F
NH
F
Celecoxib
Sulphamethoxazole
Fig. 1. Biologically active drugs having Sulfonamide and Schiff base moieties.
This research work leads to the synthesis of compounds having sulfonamide and Schiff base
moieties. Sulfonamides are widely used as antimicrobial [2, 3], anticancer [4, 5], anti-
inflammatory [6] and antiviral agents as well as HIV protease inhibitors [7]. Some sulfonamide
derivatives are also well recognized as an antimetabolite [8]. Sulfonamides have also shown very
good cytotoxic effects against breast cancer cells [9]. Sulfonamides were the first effective

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chemotherapeutic agents which were used competently to check and treat the bacterial infection
in human beings [10-13]. Clinically sulfonamides are used to treat several urinary tract infections
and gastrointestinal infections [14]. Some sulfonamide derivatives were screened for their
antioxidant activity, which showed good results [15]. Sulfonamide being very good
phamacophore have also shown very good inhibition against AChE and BChE enzymes [16-18].
Schiff bases also form a group of very important compounds having applications in biological,
industrial, pharmaceutical and many other fields of science. Schiff bases have antibacterial,
antifungal [19-21], and anti-tumor activity [22]. Schiff base derivatives of different compounds
which can act as anticancer and anti HIV agents have attracted the attention of modern
researchers [23]. These are also used for the treatment of mental disorders and leprosy [24].
By keeping in mind the immense biological significance of sulfonamides and Schiff bases,
sulfonamide hybrid Schiff bases (3-20) were synthesized and further investigated their
antioxidant and enzyme inhibition potential against AChE and BChE enzymes. Likewise, to
develop structure activity relationship (SAR) for these synthesized compounds, the effects of
various substituents on the enzyme inhibition activity were studied.
2. Materials and Methods
Analytical grade Hydrazine hydrate, 4-chlorobenzenesulfonyl chloride, methyl anthranilate and
13
aldehydes were obtained from Aldrich (USA). NMR (1H and C) were noted using JEOL- 500
MHz DELTA2_NMR Spectrometer using DMSO-d6 as solvent. The selected single crystal was
analyzed by Agilent Technologies Diffractometer (Agilent SuperNova-Dual source). For data
collection, it is equipped with micro-focus Cu/Mo Kα radiation. The data collection was
accomplished using CrysAlisPro software at 296 K. SHELXS–97 methods was used for structure
solution and refinement [25]. PLATON and ORTEP [26] in built with WinGX were used for
figures generation.
All the X-H (X = C, N and O) hydrogen atoms were positioned geometrically and treated as
riding atoms with C–H = 0.93 Å and Uiso(H) = 1.2 Ueq(C) for aromatic carbon atoms. The
methyl hydrogens were positioned geometrically with Cmethyl-H = 0.96 Å and Uiso(H) = 1.5
Ueq(C). The N-H and O-H hydrogen with N-H = 0.83 (3) - 0.92 (3) Å, O-H = 0.76 Å were

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refined using riding model with Uiso (H) = 1.2 Ueq(N) and Uiso (H) = 1.5 Ueq(O). The Crystal
data was placed at the Cambridge Crystallographic Data Centre. The deposition number which is
assigned to it is 1866050. This is known as CCDC number for molecule (09). This Crystal data
can be obtained without any charges on application to CCDC 12 Union Road, Cambridge CB21
EZ, UK. (Fax: (+44) 1223 336-033; e-mail: data_request@ccdc.cam.ac.uk).
2.1. Synthesis of methyl 2-{[(4-chlorophenyl)sulfonyl]amino}benzoate (01)
Weighed amount of methyl anthranilate was dissolved in pyridine (10 mL). Solution of 4-
chlorobenzenesulphonyl chloride in pyridine was added into the solution of methyl anthranilate.
Reaction mixture was allowed to stir at room temperature. The pH of this mixture was retained
up to 7 by continuous addition of sodium bicarbonate into the reaction mixture. The progress of
the reaction was monitored through thin layer chromatography. The reaction mixture was filtered
to remove excess salts. White crystals of the product appeared in the filtrate which were
collected and washed with cold methanol. (Scheme 1)
2.2. Synthesis of 4-chloro-N-[2-(hydrazinocarbonyl)phenyl]benzene sulfonamide (02)
Weighed amount of methyl 2-{[(4-chlorophenyl)sulfonyl]amino}benzoate (01) was dissolved in
acetonitrile followed by addition of excess hydrazine and refluxed the mixture for two hours.
After the completion of the reaction, acetonitrile was removed under vacuum. Extra hydrazine
was removed by performing ethyl acetate /water extraction. Ethyl acetate layer was evaporated
under vacuum to get the pure product as white solid. The product was recrystallized in ethanol to
get colorless crystals of the product. (Scheme 1)
2.3. General procedure for the synthesis of Schiff bases of 4-chloro-N-[2-(hydrazinocarbonyl)
phenyl]benzenesulfonamide (03-20)
Stoichiometric amounts of different aldehydes and 4-chloro-N-[2-(hydrazinocarbonyl)
phenyl]benzenesulfonamide (02) were dissolved in methanol (15 mL). The mixtures were
allowed to reflux for 2 hours. The progress of the reactions was monitored using thin layer
chromatography. Upon completion of reactions, solvents were evaporated under reduced
pressure. The products were washed with cold ethanol and recrystallized by methanol to obtain
the products in good yield (above 90%). (Scheme 1)

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O
O
CH3
O
O
S
Cl
H2N
O
Cl
Pyridine
NH
+
Stirring, 2 Hrs
S
O
H3C
O
O
Cl
(01)
Cl
H2N NH2
O
O
H
S
O
R3
HN
R1
R2
N
NH
O
NH2
NH
R2
R1
O
Cl
(03-19)
R3
Cl
NH
S
O
O
O
O
S
(02)
O
HN
N
NH
(20)
O
Where
Compounds
R1
H
R2
R3
Cl
Br
H
H
H
H
H
H
H
H
H
03
04
05
06
07
08
09
10
11
H
H
N(CH3)2
H
H
H
NO2
OH
H
OH
OCH3
CH(CH3)2
CH3
H
CH3

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OH
CF3
H
H
H
H
H
12
13
14
15
16
17
18
19
H
CF3
H
H
F
H
H
F
H
H
COOH
H
COOH
H
H
OCH3
OH
Scheme 1. Synthesis of methyl 2-{[(4-chlorophenyl)sulfonyl]amino}benzoate (01) and 4-chloro-
N-[2-(hydrazinocarbonyl)phenyl]benzenesulfonamide (02) and their Schiff base derivatives (03-
20).
2.4. Characterization
The spectral data for all the synthesized compounds is represented here
Methyl 2-{[(4-chlorophenyl)sulfonyl]amino}benzoate (01)
Yield: 81.3%; M.F: C14H12ClNO4S; M.P: 97±1°C; FTIR (KBr pellet, cm−1): 1255.22 (C-O,
stretch), 1346.19 (S=O, stretch), 1676.95 (ester C=O, stretch), 3133.49 (NH, stretch). PNMR
(300MHz, DMSO-d6: δ = 3.67 (3H, s, CH3O), 7.07-7.72 (8H, m, Aromatic CH), 10.15 (1H, s,
NH-SO2). 13C NMR (DMSO-d6): 51.1(CH3O), 110.1 - 147.9(benzene ring), 168.5(C=O). MS
(ESI), 70 eV: m/z (%), 324 (100, [M] -.), 310 (10), 266 (25), 175 (25).
4-Chloro-N-[2-(hydrazinocarbonyl)phenyl] benzene sulfonamide (02)
Yield: 70.4%; M.F: C13H12ClN3O3S; M.P: 157±1°C; FTIR (KBr pellet, cm−1): 1337.86 (S=O,
stretch), 1630.37 (C=O, stretch), 3272.20 (SO2N-H, stretch), 3334.70 (CON-H, Stretch), 3390.13
(NH2 sym. stretch), 3556.32 (NH2 anti-sym. stretch). PNMR (300 MHz, DMSO- d6: δ = 4.77
(2H, NH2, amine), 7.12-7.76 (8H, m, Aromatic CH), 10.10 (1H, s, NH-SO2), 11.60 (1H, NH-
13
NH2). C NMR (DMSO-d6): 116.4 - 145.5, (benzene rings), 164.9 (NHCO). MS (ESI), 70 eV:
m/z (%), 324(100, [M] -.), 310 (10), 266 (20), 175 (20).

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4-Chloro-N-[2-({(2E)-2-[(4-chlorophenyl)methylidene]hydrazino}carbonyl)phenyl]benzene
sulfonamide (03)
Yield: 85%; M.F: C20H15Cl2N3O3S; M.P: 201-202°C; FTIR (KBr pellet, cm−1): 1336.95 (S=O,
stretch), 1599.56 (N=CH, Stretch), 1651.94 (C=O, stretch), 3319.65 (NH, stretch). PNMR (300
M Hz, DMSO- d6: δ=7.35 -7.79 (12H, m, Aromatic CH), 8.15 (1H, s, N=CH), 10.16 (1H, s, NH-
SO2), 11.44 (1H, s, NH-N). 13C NMR (DMSO-d6): 116.8 - 146.7, (benzene ring), 142.5 (CH=N),
162.9 (NHCO). MS (ESI), 70 eV: m/z (%), 446 (100, [M] -.), 418 (05), 309 (10), 182 (05).
N-[2-({(2E)-2-[(4-bromophenyl)methylidene]hydrazino}carbonyl)phenyl]-4-chlorobenzene
sulfonamide (04)
Yield: 87.1%; M.F: C20H15ClBrN3O3S; M.P: 208-209°C; FTIR (KBr pellet, cm−1): 1333.15
(S=O, stretch), 1590.20 (N=CH, Stretch), 1646.50 (C=O, stretch), 3335.65 (NH, stretch). PNMR
(300 MHz, DMSO-d6: δ = 7.24-7.81 (12H, m, Aromatic CH), 8.25 (1H, s, N=CH), 10.37 (1H, s,
NH-SO2), 11.40 (1H, s, NH-N). 13C NMR (DMSO-d6): 121.7 - 141.8 (benzene ring), 142.6
(CH=N), 163.7 (NHCO). MS (ESI), 70 eV : m/z (%), 491.02 (100), [M]-.), 464 (10), 311 (10),
268 (05).
4-Chloro-N-[2-({(2E)-2-[(4-N, N-dimethylaminophenyl)methylidene]hydrazino}carbonyl)
phenyl] benzenesulfonamide (05)
Yield: 86%; M.F: C22H21ClN4O3S; M.P: 147 °C; FTIR (KBr pellet, cm−1): 1338.44 (S=O,
stretch), 1594.03 (N=CH, Stretch), 1631.74 (C=O, stretch), 3202.25 (NH, stretch). PNMR ( 300
MHz, DMSO-d6 :δ= 2.85 (6H, s, 2CH3, CH3-N), 6.78-7.83 (12H, m, Aromatic CH), 8.35 (1H, s,
N=CH), 10.78 (1H, s, NH-SO2), 11.83 (1H, s, NH-N). 13C NMR (DMSO-d6): 40.3 (CH3N),
114.5 - 151.7 (benzene ring), 143.6 (CH=N), 163.7 (NHCO). MS (ESI), 70 eV : m/z (%), 447
(100), [M]-.), 428 (10), 309 (10), 266 (05).
4-Chloro-N-(2-{[(2E)-2-(phenylmethylidene)hydrazino]carbonyl}phenyl)benzenesulfonamide(6)
Yield: 81%; M.F: C20H16ClN3O3S; M.P: 145-146°C; FTIR (KBr pellet, cm−1): 1337.75 (S=O,
stretch), 1601.89 (N=CH, Stretch), 1646.95 (C=O, stretch), 3362.50 (NH, stretch). PNMR (300
MHz, DMSO-d6: δ = 7.19-7.86 (13H, m, Aromatic CH), 8.08 (1H, s, N=CH), 10.14 (1H, s, NH-

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SO2), 11.43 (1H, s, NH-N). 13C NMR (DMSO-d6): 115.7 - 144.9 (benzene ring), 140.9 (CH=N),
163.2 (NHCO). MS (ESI), 70 eV : m/z (%), 412.25 (100), [M]-.), 384.42 (10), 370.08 (10), 309
(10), 266(12).
4-Chloro-N-[2-({(2E)-2-[(4-nitrophenyl)methylidene]hydrazino}carbonyl)phenyl]benzene
sulfonamide (07)
Yield: 88%; M.F: C20H15ClN4O5S; M.P: 250-251°C; FTIR (KBr pellet, cm−1): 1334.93 (S=O,
stretch), 1595.43(N=CH, Stretch), 1656.36 (C=O, stretch), 3313.90 (NH, stretch). PNMR (300
MHz, DMSO- d6: δ = 7.23-8.27 (12H, m, Aromatic CH), 8.51 (1H, s, N=CH), 10.49 (1H, s, NH-
SO2), 11.57 (1H, s, NH-N).13C NMR (DMSO-d6: 163.1 (NHCO), 115.8 - 150.7 (benzene ring),
141.9(CH=N), 163.2 (NHCO). MS (ESI), 70 eV : m/z (%), 457.7 (15), [M]-.), 430.8 (60), 309
(12), 266 (05).
4-Chloro-N-[2-({(2E)-2-[(2,4-dihydroxyphenyl)methylidene]hydrazino}carbonyl)phenyl]
benzenesulfonamide (08)
Yield: 76.7%; M.F: C20H16ClN3O5S; M.P: 264-265°C; FTIR (KBr pellet, cm−1): 1323.98 (S=O,
stretch), 1605.32 (N=CH, Stretch), 1652.85 (C=O, stretch), 3286.95 (NH, stretch), 3460.41 (OH,
stretch). PNMR(300 MHz, DMSO- d6: δ= 6.19-7.84 (11H, m, Aromatic CH), 8.67 (1H,s, N=CH
), 10.87 (1H, s, NH-SO2), 11.16 (2H, s, 2OH), 12.14 (1H, s, NH-N). 13C NMR (DMSO-d6):
116.1 - 162.2 (benzene rings), 142.0 ( CH=N), 163.1 (NHCO). MS (ESI), 70 eV: m/z (%),
444.67 (15, [M]-.), 416.86 (10), 309 (13), 266 (05).
4-Chloro-N-[2-({(2E)-2-[(4-methoxyphenyl)methylidene]hydrazino}carbonyl)phenyl]benzene
sulfonamide (09)
Yield: 70%; M.F: C21H18ClN3O4S; M.P: 142°C; FTIR (KBr pellet, cm−1): 1337.05 (S=O,
stretch), 1599.07 (N=CH, Stretch), 1641.85 (C=O, stretch), 3197.69 (NH, stretch). PNMR(300
MHz, DMSO- d6: δ= 3.82 (3H, s, OCH3), 6.97-7.70 (12H, m, Aromatic CH), 8.30 (1H, s,
N=CH), 10.80 (1H, s, NH-SO2), 11.73 (1H, s, NH-N). 13C NMR (DMSO-d6): 55.9 (CH3O),
116.3 - 163.0 (benzene rings), 144.7 (CH=N), 163.1 (NHCO). MS (ESI), 70 eV : m/z (%), 442
(100), [M]-.), 310 (10), 265 (05).

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4-Chloro-N-[2-({(2E)-2-[(4-isopropylphenyl)methylidene]hydrazino}carbonyl)phenyl]benzene
sulfonamide (10)
Yield: 71.3%; M.F: C23H22ClN3O3S; M.P: 173-174°C; FTIR (KBr pellet, cm−1): 1340.25 (S=O,
stretch), 1595.64 (N=CH, Stretch), 1631.90 (C=O, stretch), 3213.31 (NH, stretch). PNMR (300
MHz, DMSO- d6: δ = 1.29 ( 6H, d, 2CH3-CH), 3.12 (1H, sep, CH-CH3), 7.22-7.86 (12H, m,
Aromatic CH), 8.29 (1H, s, N=CH), 10.23 (1H, s, NH-SO2), 11.38 (1H, s, NH-N). 13C NMR
(DMSO-d6): 23.4 (2CH3C), 36.3 (CH-CH3), 116.7 - 150.9 (benzene rings), 141.0 (CH=N), 163.3
(NHCO). MS (ESI), 70 eV: m/z (%), 454.8 (100), [M] +.), 426.94 (20), 311 (10), 266 (05).
4-chloro-N-[2-({(2E)-2-[(2,4-dimethylphenyl)methylidene]hydrazino}carbonyl)phenyl]benzene
sulfonamide(11)
Yield: 80.4%; M.F: C22H20ClN3O3S; M.P: 216-217 °C; FTIR (KBr pellet, cm−1): 1337.63 (S=O,
stretch), 1593.45 (N=CH, Stretch), 1625.73 (C=O, stretch), 3179.77 (HN, stretch). PNMR (300
MHz, DMSO- d6: δ = 2.35 ( 6H, s, 2CH3), 6.99-7.75 (11H, m, Aromatic CH), 8.31 (1H, s,
N=CH), 10.11 (1H, s, NH-SO2), 11.49 (1H, s, NH-N). 13C NMR (DMSO-d6): 18.2-24.6 (2CH3),
115.9 - 145.5 (benzene rings), 142.6 (CH=N), 163.1 (NHCO). MS (ESI), 70 eV : m/z (%), 441
(100), [M]-.), 413 (10), 224 (12), 309 (15), 266 (10).
4-chloro-N-[2-({(2E)-2-[(2-hydroxyphenyl)methylidene]hydrazino}carbonyl)phenyl]benzene
sulfonamide (12)
Yield: 70.25%; M.F: C20H16ClN3O4S; M.P: 223-224 °C; FTIR (KBr pellet, cm−1): 1335.82
(S=O, stretch), 1609.25 (N=CH, Stretch), 1639.28 (C=O, stretch), 3271.79 (HN, stretch),
3341.66 (OH, stretch). PNMR (300 MHz, DMSO- d6: δ = 6.92-7.71 (12H, m, Aromatic CH),
8.58 (1H, s, N=CH), 10.75 (1H, s, NH-SO2), 11.10 (1H, s, 1OH), 12.10 (1H, s, NH-N). 13C NMR
(DMSO-d6): 116.4 - 162.2 (benzene rings), 141.8 (CH=N), 162.6 (NHCO). MS (ESI), 70 eV :
m/z (%), 428.88 (100), [M]-.), 401 (10), 211 (15), 309 (20), 267 (100).
4-chloro-N-(2-{[(2E)-2-{[2-(trifluoromethyl)phenyl]methylidene}hydrazino]carbonyl}phenyl)
benzenesulfonamide (13)
Yield: 78.4%; M.F: C21H15ClF3N3O3S; M.P: 224 °C; FTIR (KBr pellet, cm−1): 1335.52 (S=O,
stretch), 1628.95 (C=O, stretch), 1597.50 (N=CH, Stretch), 3198.39 (HN, stretch). PNMR (300

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MHz, DMSO- d6: δ = 6.98 - 7.78 (12H, m, Aromatic CH), 8.39 (1H, s, N=CH), 10.19 (1H, s,
NH-SO2), 11.46 (1H, s, NH-N). 13C NMR (DMSO- d6): 119.7 – 142.7 (benzene rings), 114.5
(1CF3), 142.2 (CH=N), 161.9 (NHCO). MS (ESI), 70 eV : m/z (%), 480.88 (100), [M]-.), 453
(10), 264 (20), 309 (15), 266 (10).
4-chloro-N-(2-{[(2E)-2-{[4-(trifluoromethyl)phenyl]methylidene}hydrazino]carbonyl}phenyl)
benzenesulfonamide (14)
Yield: 77.3%; M.F: C21H15ClF3N3O3S; M.P: 193 °C; FTIR (KBr pellet, cm−1): 1323.56 (S=O,
stretch), 1603.45 (N=CH, Stretch), 1659.60 (C=O, stretch), 3325.42 (HN, stretch). PNMR (300
MHz, DMSO- d6: δ = 6.87 - 7.79 (12H, m, Aromatic CH), 8.26 (1H, s, N=CH), 10.18 (1H, s,
NH-SO2), 11.47 (1H, s, NH-N). 13C NMR (DMSO-d6): 112.6 - 146.5 (benzene rings), 120.7
(CF3), 140.8 (CH=N), 162.5 (NHCO). MS (ESI), 70 eV : m/z (%), 480.25 (100), [M]-.), 456.08
(15), 412.17 (10), 324.17 (15).
4-chloro-N-[2-({(2E)-2-[(3-fluorophenyl)methylidene]hydrazino}carbonyl)phenyl]benzene
sulfonamide (15)
Yield: 81.4%; M.F: C20H15ClFN3O3S; M.P: 196 °C; FTIR (KBr pellet, cm−1): 1333.42 (S=O,
stretch), 1597.50 (N=CH, Stretch), 1640.80 (C=O, stretch), 3334.96 (HN, stretch). PNMR (300
MHz, DMSO- d6: δ = 6.80-7.94 (12H, m, Aromatic CH), 8.22 (1H, s, N=CH), 10.35 (1H, s, NH-
SO2), 11.44 (1H, s, NH-N). 13C NMR (DMSO-d6): 113.6 - 160.5 (benzene rings), 141.2 (CH=N),
164.1 (NHCO). MS (ESI), 70 eV : m/z (%), 430.01(100), [M] -.).
4-chloro-N-[2-({(2E)-2-[(4-fluorophenyl)methylidene]hydrazino}carbonyl)phenyl]benzene
sulfonamide (16)
Yield: 80.4%; M.F: C20H15ClFN3O3S; M.P: 187 °C; FTIR (KBr pellet, cm−1): 1334.25 (S=O,
stretch), 1602.25 (N=CH, Stretch), 1654.36 (C=O, stretch), 3323.28 (HN, stretch). PNMR (300
MHz, DMSO-d6: δ = 6.89 - 7.80 (12H, m, Aromatic CH), 8.13 (1H, s, N=CH), 10.11 (1H, s,
NH-SO2), 11.45 (1H, s, NH-N). 13C NMR (DMSO-d6): 117.1 - 163.6 (benzene rings), 144.1
(CH=N), 162.8 (NHCO). MS (ESI), 70 eV : m/z (%), 430.02(100), [M]+.).
4-[(E)-{[(2-{[(4-chlorophenyl)sulfonyl]amino}phenyl)carbonyl]hydrazono}methyl]benzoic acid
(17)

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Yield: 80.4%; M.F: C21H16ClN3O5S; M.P: 267 °C; FTIR (KBr pellet, cm−1): 1343.56 (S=O,
stretch), 1612.69 (N=CH, Stretch), 1654.23 (C=O, stretch), 1680.65 (carboxyl C=O, stretch),
3100.28 (carboxyl OH, stretch), 3269.36 (HN, stretch). PNMR (300 MHz, DMSO- d6: δ = 6.89 -
8.14 (12H, m, Aromatic CH), 8.34 (1H, s, N=CH), 10.20 (1H, s, NH-SO2), 11.05 (1H, s,
COOH), 11.71 (1H, s, NH-N). 13C NMR ( DMSO- d6): 115.6 - 147.2 (benzene rings), 142.7
(CH=N), 165.1 (NHCO), 173.2 (COOH). MS (ESI), 70 eV : m/z (%), 456.89 (100), [M] -.), 429
(15), 240 (10).
2-[(E)-{[(2-{[(4-chlorophenyl)sulfonyl]amino}phenyl)carbonyl]hydrazono}methyl]benzoic acid
(18)
Yield: 80.4%; M.F: C21H16ClN3O5S; M.P: 234 °C; FTIR (KBr pellet, cm−1): 1340.23 (S=O,
stretch), 1601.50 (N=CH, Stretch), 1634.84 (C=O, stretch), 1712.18 (carboxyl C=O, stretch),
3182.88 (carboxyl OH, stretch), 3301.75 (HN, stretch). PNMR (300 MHz, DMSO-d6: δ = 6.99-
7.79 (12H, m, Aromatic CH), 8.15 (1H, s, N=CH), 10.09 (1H, s, NH-SO2), 11.12 (1H, s,
COOH), 11.69 (1H, s, NH-N). 13C NMR (DMSO-d6): 119.3 - 143.8 (benzene rings), 147.5
(CH=N), 163.1 (NHCO), 169.4 ( COOH). MS (ESI), 70 eV : m/z (%), 456.89 (100), [M]-.), 429
(10), 240 (15).
4-chloro-N-[2-({(2E)-2-[(4-hydroxy-3-methoxyphenyl)methylidene]hydrazino}carbonyl)phenyl]
benzenesulfonamide (19)
Yield: 80.4%; M.F: C21H18ClN3O5S; M.P: 229 °C; FTIR (KBr pellet, cm−1): 1339.60 (S=O,
stretch), 1586.37 (N=CH, Stretch), 1630.21 (C=O, stretch), 3181.95 (HN, stretch), 3455.80 (OH,
stretch). PNMR (300 MHz, DMSO- d6: δ = 3.78 (3H, s, OCH3), 6.61-7.89 (11H, m, Aromatic
13
CH), 8.31 (1H, s, N=CH), 10.63 (1H, s, NH-SO2), 11.09 (1H, s, OH), 11.98 (1H, s, NH-N). C
NMR (DMSO-d6): 56.3 (OCH3), 116.7 - 151.5 (benzene rings), 144.6 (CH=N), 163.2 (NHCO).
MS (ESI), 70 eV : m/z (%), 459 (15), [M] -.), 431 (05), 242 (10), 311 (15).
4-Chloro-N-[2-({(2E)-2-[(2E)-3-phenylprop-2-en-1-ylidene]hydrazino}carbonyl)phenyl]benzene
sulfonamide (20)
Yield: 70.4%; M.F: C22H18ClN3O3S; M.P: 199 °C; FTIR (KBr pellet, cm−1): 1340.09 (S=O,
stretch), 1599.02 (N=CH, Stretch), 1643.03 (C=O, stretch), 3240.15 (HN, stretch). PNMR (300
MHz, DMSO-d6: δ = 7.28-7.69 (13H, m, Aromatic CH), 7.89 (1H, s, N=CH), 10.71 (1H, s, NH-

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SO2), 11.94 (1H, s, NH-N). 13C NMR (DMSO-d6): 116.7 - 141.6 (benzene rings), 135.8 (CH=N),
137.9 (CH=CH), 165.18 (NHCO). MS (ESI), 70 eV : m/z (%), 438.8 (15), [M]+.), 411 (20), 309
(10), 266 (15).
2.5. Biological activity
2.5.1. Enzyme Inhibition Essay
Ryan and Elman method [27] was used to evaluate enzyme inhibition with little modification.
50 ꢀL of the enzyme (AChE/BChE) was taken and mixed with 50 ꢀL of each sample (50ꢀM)
(01-20). The mixture of each sample was allowed to stand for 10 minutes. Then 50 ꢀL of
substrate i.e. butyrylthiocholine chloride (0.2mM) for BChE and acetylthiocholine iodide(0.71
mM) for AChE was added to each sample along with 50 ꢀL(0.5 mM) of DTNB [5,5´-dithio-
bis(2-nitrobenzoic acid)] and 500 ꢀL of phosphate buffer at pH 8 and shaked well. This mixture
was then incubated at 37 °C for 30 minutes. 5-thio-2- nitrobenzoate anion was formed due to the
hydrolysis of the substrate which gave the yellow coloration of the mixture. Spectrophotometric
method was used to monitor the yellow coloration produced due to the hydrolysis of the
substrate by measuring absorbance at 412 nm and 400 nm for BChE and AChE, respectively.
Following formula was used to calculate percentage enzyme inhibition for all the samples.
ꢊ − ꢋ
%ꢀꢁꢂ ꢃꢄℎꢅꢆꢅꢇꢅꢈꢄ = ꢉ
ꢌ × 100
ꢊ
Where A =Absorption value of enzyme having test compounds
B = Absorption value of the enzyme without any test compounds
Every experiment was performed three times and average value was utilized. Donepzil was used
as reference drug.
2.5.2. Antioxidant assay
DPPH scavenging methodology [28] was utilized to find the antioxidant potential of the
synthesized compounds (01-20). 2 mL of 0.2mM ethanolic solution of DPPH was taken and 100
uL of 50 uM solution of the samples was added into it. Incubation of the mixture was done at 37

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oC for 20 minutes. Then Spectrophotometric method was used to find the disappearance of
DPPH coloration by taking absorbance at λ = 516 nm. 1mM ascorbic acid was utilized as
positive control. Following equation was used to calculate the free radical scavenging (%age) for
all the samples
ꢋ° − ꢋꢐ
%ꢀꢁꢂ ꢍꢎꢀꢏꢂꢄꢁꢅꢄꢁ = ꢉ
ꢌ × 100
ꢋ°
Where
AT = Absorbance of the sample solution,
Ao = Absorbance of the DPPH solution,
2.6. Molecular Docking studies
Molecular docking studies were carried out to check enzyme and ligand interactions. Crystal
structure of enzymes AChE and BChE was acquired from (RCSB) protein data bank. ACD
Chem sketch software was utilized to sketch the molecular structures of the ligands and 3D Pro
12.0 software was utilized to obtain 3D optimization of the ligands and SYBYL mol2 file format
was used to save the optimized structures. Process of docking was performed using Auto Dock
Tool v 1.5.6., 100 different configurations were attained. One of the best conformations of the
compound was selected after visualizing all the conformations using Discovery Studio Visualizer
4.0.
3. Results and Discussion
3.1. Chemistry
Sulfonamide based Schiff bases of anthranilic acid were synthesized according to Scheme 1.
Their structures were characterized by FTIR, 1H NMR, 13C NMR and X-ray-Crystallography.
3.1.1. FT-IR studies
Functional groups identification of all the synthesized compounds was done using FTIR spectra.
The presence of imine linkage was confirmed with the peaks in the range of ῡ = 1586.37 cm-1-
1612.69 cm-1 (N=CH) and 1625.73 cm-1- 1656.36 cm-1 (C=O). CH aromatic stretching vibrations
lies in the region 3100-3040 and other CH peaks in the region 2940-2800 cm-1.

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3.1.2. 1H NMR Studies
1H NMR spectra of synthesized compounds confirmed their structures. Compound (01) was
converted to compound (02), peak of OCH3 at 3.67ppm disappeared and new peaks appeared at δ
= 4.77ppm, corresponding to NH2. Similarly when compound (02) was further converted to its
Schiff base derivatives (03-20), peaks appeared in the range of δ = 7.89ppm to 8.58ppm
corresponding to their N=CH bond. Peaks corresponding to aromatic protons were appeared
between 6.19ppm - 8.27ppm, depending upon the type of substituents attached to aromatic ring.
Electron donating effects of dimethyl amino, hydroxyl and methoxyl groups increased the
electron density at ortho and para positions that caused the shifting of the peak of the
corresponding H to field up (6.19ppm - 6.92ppm) in compounds (05, 08, 12, 09 and 19) as
compared to 7.19ppm values protons of non-substituted ring (compound 06). In contrast,
electron withdrawing group (NO2 group) caused the shifting of chemical shifts downfield (higher
ppm values up to 8.27ppm in compound 07. In compounds 17 and 18, signal of carboxyl
hydrogen appear at 11.05ppm – 11.12ppm, because carboxyl proton is highly de-shielded. In
compounds 09 and 19, peaks appear in the range of δ = 3.78ppm - 3.82ppm corresponding to
protons of methoxyl groups. All the spectral data for Proton NMR are in good agreement with
the structures of the synthesized compounds.
3.1.3. 13C NMR studies
13C NMR spectra also supported the molecular structures of the series. Peak of carbonyl carbon
(C=O) of ester group at 168.5ppm in compound (01) has been shifted towards lower ppm values
at 164.9ppm in compound (02) due to the formation of amide linkage, which is in accordance
with the literature values. In compounds (03-20), peak for imine carbon (N=C-H) appeared in the
range of 135.8ppm – 147.5ppm. Variation of the peaks of aromatic carbons in the range of
114.5ppm - 169.5ppm depending upon the nature of substituents is also in accordance with the
structures of the synthesized compounds as described in the spectral data in experimental section.
3.1.4. Mass spectroscopic studies
The molecular masses of the synthesized compounds were established by Electron Spray
Ionization (ESI) technique. The mass spectrum of all the compounds showed characteristic peaks
corresponding to [M-H] ions in negative mode and [M+H] peaks in positive mode. The

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compounds (2-20) contain chlorine atom which is confirmed by its [M+2] isotopic peak (33%).
While in compound 03, that [M+2] peak was enhanced up to 66% due to the presence of two
chlorine atoms. All the spectral data for MS (ESI) are in good accordance with the structures of
the synthesized compounds.
3.1.5. XRD studies
The molecules in the unit cell of the target compound (09) arranged themselves to generate the
monoclinic crystal system with one molecule in an asymmetric unit cell and a methanol molecule
as solvent as shown in Fig. 2 (a). The crystallographic parameters are given in Table 1 while
selected bond lengths and bond angles are provided in Tables 2 and 3. The geometry around the
central S atom of sulfonamide group adopted the distorted tetrahedral geometry [29-32]. There
are the three aromatic rings in the molecule. The central aromatic ring (C1-C6) is oriented at
dihedral angle of 54.227 (7)° and 46.482 (8)° with respect to the 4-chlorophenyl (C7-C12) and 4-
methoxyphenyl (C15-C20) aromatic rings. The donor-nitrogen atom N1 at the position (x, y, z)
connects with the acceptor atom O3 via intra-molecular hydrogen bonding interaction and
produced six membered ring motifs as shown in Fig. 2 (b) [33]. The root mean square deviation
for this ring (C1/C6/C13/N1/H1N/O3) is 0.1129(3)° where the most deviations were observed
from the C13 = 0.11861 Å and O3 = 0.1611Å atoms. This ring is twisted with the dihedral angle
of 9.411(3)° with its fused ring (C1-C6) and its dihedral angle with the 4-methoxyphenyl (C15-
C20) aromatic ring is 50.001 (3)°. The N2-H2N...O5 interaction along-with O5-H1O...O3 type
intermolecular hydrogen bonding connects the molecules along a-axis to generate the infinite
long chains as shown in Fig. 3 (a). Another non-classical interaction, in which C11 acts as
hydrogen atom donor and O2 acts as acceptor atom following the symmetry operation -x, y+1/2,
-z+3/2, connects the chains and give rise to formation of two-dimensional network along ab-
plane as shown in Fig. 3 (b) and Table 4.

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Fig. 2. a) ORTEP diagram of compound 09, thermal ellipsoids were drawn at 50% probability
level. b) A unit cell view showing the intra- and inter-molecular hydrogen bonding interactions
in compound 09.
Fig. 3. a)Formation of long chains along the diagonal of b-axes for compound 09. b) A view
showing the long chain generation along a-axis for compound 09.
Table 1
Crystal data and structure refinement for compound 09

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Crystal data and structure refinement for compound 09
CCDC number
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
1866050
C22H22ClN3O5S
475.93
296(2)
monoclinic
P21/c
a/Å
6.6203(5)
b/Å
15.8815(15)
c/Å
22.143(2)
α/°
90
β/°
97.386(8)
γ/°
90
2308.8(4)
Volume/Å3
Z
4
1.369
0.294
ρcalc mg/mm3
ꢀ/mm-1
F(000)
992.0
Crystal size/mm3
2θ range for data collection
Index ranges
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
Final R indexes [I>=2σ (I)]
Final R indexes [all data]
Largest diff. peak/hole / e Å-3
0.48 × 0.42 × 0.33
6.128 to 58.134°
-8 ≤ h ≤ 8, -21 ≤ k ≤ 11, -29 ≤ l ≤ 29
12501
5462[R(int) = 0.0446]
5462/0/299
1.018
R1 = 0.0597, wR2 = 0.1336
R1 = 0.1243, wR2 = 0.1701
0.21/-0.34
Table 2
Bond Lengths for compound 09.
AtomAtom Length/Å AtomAtom Length/Å
C1 C2
C1 C6
C1 N1
C2 C3
1.379(4)
1.405(4)
1.434(4)
1.377(4)
C13 O3
C14 C15
C14 N3
C15 C16
1.243(3)
1.439(4)
1.276(4)
1.398(4)

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C3 C4
C4 C5
C5 C6
C6 C13
C7 C8
C7 C12
C7 S1
C8 C9
C9 C10
C10 C11
C10 Cl1
C11 C12
C13 N2
1.369(5)
1.373(4)
1.395(4)
1.479(4)
1.384(4)
1.376(4)
1.759(3)
1.374(5)
1.363(5)
1.378(5)
1.735(3)
1.376(5)
1.341(4)
C15 C20
C16 C17
C17 C18
C18 C19
C18 O4
C19 C20
C21 O4
C22 O5
N1 S1
N2 N3
O1 S1
O2 S1
1.389(4)
1.370(4)
1.387(4)
1.374(4)
1.363(4)
1.374(4)
1.416(4)
1.379(5)
1.640(3)
1.387(3)
1.420(2)
1.429(2)
Table 3
Bond Angles for compound 09.
AtomAtomAtom
Angle/˚
AtomAtomAtom
Angle/˚
C2 C1 C6
C2 C1 N1
C6 C1 N1
C3 C2 C1
C4 C3 C2
C3 C4 C5
C4 C5 C6
C1 C6 C13
C5 C6 C1
C5 C6 C13
C8 C7 S1
C12 C7 C8
C12 C7 S1
C9 C8 C7
C10 C9 C8
C9 C10 C11
C9 C10 Cl1
C11 C10 Cl1
C12 C11 C10
C7 C12 C11
N2 C13 C6
120.1(3)
118.5(3)
121.4(2)
120.4(3)
120.5(3)
119.7(3)
121.4(3)
121.1(2)
117.9(3)
121.0(3)
120.1(2)
120.4(3)
119.4(2)
119.7(3)
119.4(3)
121.6(3)
119.2(3)
119.1(3)
119.0(3)
119.8(3)
115.8(2)
O3 C13 N2
N3 C14 C15
C16 C15 C14
C20 C15 C14
C20 C15 C16
C17 C16 C15
C16 C17 C18
C19 C18 C17
O4 C18 C17
O4 C18 C19
C18 C19 C20
C19 C20 C15
C1 N1 S1
C13 N2 N3
C14 N3 N2
C18 O4 C21
N1 S1 C7
O1 S1 C7
O1 S1 N1
O1 S1 O2
O2 S1 C7
122.0(3)
123.9(3)
123.7(3)
119.2(3)
117.0(3)
120.9(3)
120.6(3)
119.7(3)
115.7(3)
124.6(3)
119.3(3)
122.5(3)
119.82(19)
119.4(2)
115.0(2)
117.9(3)
104.99(14)
109.40(15)
105.09(14)
120.28(14)
108.23(14)

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O3 C13 C6
122.2(3)
O2 S1 N1
107.80(14)
Table 4
Hydrogen bond interactions in compound 09
D
H
A
d(D-H)/Å
d(H-A)/Å
d(D-A)/Å
D-H-A/°
C11 H11 O21
C14 H14 O5
N1 H1 O3
N2 H2A O5
O5 H5AO32
0.93
0.93
0.83(3)
0.92(3)
0.76(4)
2.46
2.47
2.06(3)
1.96(3)
2.13(4)
3.391(4)
3.286(4)
2.745(3)
2.874(3)
2.853(3)
176.9
145.9
141(3)
174(3)
160(4)
11+X,1+Y,+Z; 21-X,1-Y,1-Z; 31-X,1-Y,2-Z
3.2. Antioxidant activity
Antioxidant potential of the compounds can be determined by carrying DPPH free radical
scavenging activity. This technique gives antioxidant potential information in relatively shorter
times. The compounds possessing the ability to reduce the DPPH pledge this radical to form 1,1-
diphenyl-2-picrylhydrazine [34]. Deep violet color of DPPH is due to the presence of an odd
electron in it, which results in strong absorption at 517 nm [35]. The compounds possessing
antioxidant potential cause the pairing of the odd electron of DPPH, which result in decrease in
absorption at the above said wavelength. The results are interpreted in terms of %age scavenging
activity as compared to ascorbic acid chosen as standard and are shown in Table 5. All the
compounds showed moderate antioxidant activities except 2, 8 and 19 which showed very good
antioxidant activities. These compounds showed good activities due to the availability of –NH2
and –OH groups in their structures to provide hydrogen atoms to show antioxidant potential [36].
Table 5
Percentage scavenging of compounds 01-20 at 1ug/ꢀl concentration
Percentage
scavenging
Compounds
Absorbance
1.60
0.08
21
96
01
02

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1.316
1.59
35
21
53
28
29
91
19
22
20
74
16
18
21
34
19
59
96
14
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
0.963
1.496
1.477
0.182
1.655
1.587
1.638
0.525
1.704
1.667
1.603
1.343
1.601
0.826
0.08
1.742
0.09
20
+ve Control
(Ascorbic acid)
DPPH
95.5
2.031
3.3. Enzyme inhibition studies
All the synthesized compounds (01-20) were checked in vitro for their inhibitory potential
against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes using
spectrophotometric method.
The results are interpreted as %age inhibition of the AChE and BChE using Donepzil as standard
drug and are reported in Table 6. It is clear from the results that most of the compounds showed
very good inhibition against both the enzymes AChE and BChE. The most active compound was
compound 12 which showed very good inhibition potential against both the enzymes, due to the
presence of -OH group as substituent. This -OH group forms strong H-bond associations with the
active parts of both the enzymes. Compound 02 also showed very good inhibitions against both
the enzymes due to the presence of hydrazide linkage in it, which leads to formation of strong
association with the active pockets of both the enzymes through hydrogen bonding. Compound
08 also showed very good inhibition potential against both the enzymes, AChE and BChE due to
the presence of two -OH groups as substituents which lead to increased H- bond interactions
with the active sites of the enzymes. Compound 17 also depicted very good activities due to the

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presence of –COOH group at the aromatic ring, which leaded to strong electrostatic forces of
attraction with the active sites of the enzymes. Compounds 07, 15 and 16 showed good inhibition
against BChE enzyme, due to the presence of NO2 group and F atom as substituents in the
compound. These groups also cause the formation of H-bonds with the active sites of the
enzymes. Overall, most of the compounds showed good enzyme inhibition activities against both
the enzymes AChE and BChE. These outcomes have been additionally proved by In-silico
studies (molecular docking). The results depict that these sulfonamide based Schiff bases can be
further studied to develop new scaffolds to cure Alzheimer disease.
Table 6
Percentage inhibition of compounds 01-20 against AChE and BChE at 50uM concentration
Compounds
Percentage Inhibition
Percentage Inhibition
against AChE
against BChE
55
91
61
63
12
77
50
67
51
72
48
83
53
51
53
58
63
55
53
50
95
77
75
31
26
51
51
81
69
62
52
34
92
58
36
79
60
70
36
25
43
92
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
Donepzil
3.4. Molecular Docking Studies
To establish SAR studies, molecular docking studies of all the compounds (01-20) was carried
out to find protein ligand interactions by checking the binding interactions of the compound with

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the active pockets of the enzymes. To carry out molecular docking studies, Templates taken were
human BChE (PDB ID: 4BDS) and AChE (PDB ID: 4BDT) X-ray structures. After molecular
docking studies, the minimum binding energies of the compounds (01-20) calculated are given in
Table 7.
Table 7
Binding energies of selected conformations against human AChE and BChE
Compound No
h AChE Lowest Binding
h BChE Lowest
Binding Energy
∆G in kJ mol−1
-8.25
Energy ∆G in kJ mol−1
-9.06
-8.79
01
02
-8.48
-11.27
-10.41
-9.03
-9.81
-9.08
-8.43
03
04
05
-9.89
-9.47
06
-8.02
-8.98
07
-7.66
-8.65
08
-9.75
-8.81
09
-10.69
-8.97
-9.64
-9.35
10
11
-8.49
-8.39
12
-5.54
-8.67
13
-8.75
-9.44
14
-9.88
-9.41
15
-8.97
-8.80
16
-4.5
-8.18
17
-8.37
-8.25
18
19
-9.77
-8.79
-9.68
-8.77
20
−10 (HUW)
−6.83 (THA)
Standard
The molecular docking studies revealed that most stable conformations of all the compounds
showed various types of interactions with the amino acids of the active pockets of BChE and
AChE and are well accommodated there. For example visualization of the most potent
compound 12 in the active pocket of human AChE enzyme manifested many important bonding
interactions as depicted in Fig. 4. Compound 12 establishes H-bonding interactions with amino
acid residues Ser-203 utilizing –OH group of aromatic ring, Tyr-337 using sulfonamide oxygen
and aromatic ring and with His-447 through –OH group of aromatic ring. Trp-86 shows various
types of interactions with this compound including π-π-stacked interaction, π-sulfur interactions

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with sulfonamide S, as well as π-sigma interactions with aromatic ring. This compound also
shows hydrophobic π-π T-shaped interactions with PHE-338 and hydrophobic π-alkyl
interactions with Trp-439, Tyr-449, Tyr-337 and Trp-86. Moreover, amide π-stacked interactions
are being shown by this compound utilizing Gly-121 and Gly-122.
Fig. 4. Putative binding approaches of compound 12 inside AChE enzyme.
Similarly, the conception of the compound 12 in the active pocket of BChE enzyme unveiled
many significant interactions. The compound 12 forms H-bonding interactions with amino acid
residue of catalytic triad of BChE, His-438 using Hydrogen atom of Hydroxyl group and with
Thr-120 by utilizing sulfonamide oxygen atom. It also depicts π-alkyl interaction with amino
acids Trp-231 and Phe-398 using Cl atom. The catalytic amino acid Leu-286 shows hydrophobic
alkyl interactions. Π-π T-shaped interactions are shown by Trp-82 and Phe-329. Phenyl ring of
the compound 12 also showed amide-pi stacking interactions with Gly-115 and Gly-116 amino
acid residues of BChE. This compound also makes π-sigma type of interactions with amino acid
Ala-328. Glu-197 amino acid residue of BChE shows electrostatic interactions with electron
deficient nitrogen of hydrazide portion. The assumed binding type of the compound 12 can be
found in Fig. 5.

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Fig. 5. Interactions of the compound 12 with BChE at 3D space; Interactions with specific amino
acid residues are shown in the box. The 3D ribbon represents the enzyme; stick model is the
lowest energy conforms of the inhibitor 12 along with amino acids of BChE interacting with it.
Moreover, conception of the active compound 08 depicts H-bonding interactions with amino
acid residues of anionic sub site of AChE, as hydroxyl group of aromatic ring establishes H-
bonding with ‘O’ atom of Trp-86. Similarly, H-bond interactions are shown by this compound
with amino acid residues Tyr-337 using aromatic ring and Tyr-341 using sulfonamide oxygen.
This derivative shows π alkyl type interactions with Met-443, Tyr-449, Trp-439, Tyr-337, Pro-
446 and Trp-86 of AChE. Trp-86 and Trp-439 residues of AChE show π-sigma interactions with
under observed compound. Tyr-341 establishes π-sulfur interactions with sulfur atom of
sulfonamide portion of the compound. Glu-202 and Trp-86 residues of AChE show electrostatic
π- cation type interaction with partial positively charged nitrogen of hydrazide portion of the
displayed compound as portrayed in Fig. 6.

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Fig. 6. Interactions of the compound 08 with AChE at 3D space; Interactions with specific amino
acid residues are shown in the box. The 3D ribbon represents the enzyme; stick model is the
lowest energy conforms of the inhibitor 08 along with amino acids of AChE interacting with it.
The visualization of one of the compounds 08 inside active pocket of BChE enzyme revealed
many important bonding linkages. This compound develops H-bonding interactions with
catalytic amino acid residues His-438, Ser-198, Leu-286 and Gly-116. Amino acid residue Tyr-
332 also develops π-sigma interaction with this compound. This compound also forms π-π
stacked interactions with amino acid residues, His-438, Tyr-332 and Trp-82 and Phe-329 using
its aryl ring. The presumed bonding approach of this compound is presented in Fig. 7.

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Fig. 7. Interactions of the compound 08 with BChE at 3D space; Interactions with specific amino
acid residues are shown in the box. The 3D ribbon represents the enzyme; stick model is the
lowest energy conforms of the inhibitor 08 along with amino acids of BChE interacting with it.
Similarly, docking evaluation of compound 07 against AChE showed many different types of
associations. Gly-126 shows hydrogen bonding with oxygen of the nitro group. Similarly Trp-86
shows hydrogen bonding with sulfonamide oxygen atom, Tyr-337 also shows H-bonding with
aromatic ring of ligand 07. This compound also shows π-alkyl interactions with Tyr-449 and
Tyr-337. Amino acid residues Trp-439 and Tyr-337 exhibit hydrophobic π-π stacked bonding
interactions with the under study compound. Additionally, this shows π –π T-shaped interaction
with Trp-86 amino acid residue. Π-sulfur type of associations is shown by Met-443 with ligand
07. Amide π-stacked interactions are shown by Gly-120 and Gly-121 with aromatic ring of the
compound. Glu-202 and Trp-86 show electrostatic type of interactions with partially positive
nitrogen of the hydrazide part of the ligand. The bonding mode of compound 07 has been
exposed in Fig. 8.

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Fig. 8. Interactions of the compound 07 with AChE at 3D space; Interactions with specific amino
acid residues are shown in the box. The 3D ribbon represents the enzyme; stick model is the
lowest energy conforms of the inhibitor 07 along with amino acids of AChE interacting with it.
In continuation to our earlier discussion, it is found that one of the active compounds, compound
07 showed several significant interactions inside active pocket of BChE enzyme. It forms H-
bonding type interactions with amino acid residues Asp-70, Tyr-32, Ala-199, Gly-116, Gly-117
and Pro-285. π - Alkyl interactions are also shown by compound 07 with amino acid residues
His-438, Ala-328 and Trp-82. Trp-82 residue also forms π-sigma interactions with chlorine atom
attached to aromatic ring. Phe-329 and Trp-82 are also involved in π-π-T-shaped interactions. All
the interactions of compound 07 with active pocket of BChE are shown in Fig. 9.

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Fig. 9. Interactions of the compound 07 with BChE at 3D space; Interactions with specific amino
acid residues are shown in the box. The 3D ribbon represents the enzyme; stick model is the
lowest energy conforms of the inhibitor 07 along with amino acids of BChE interacting with it.
3.5. Conclusion
In conclusion, efficient synthetic method, characterization, antioxidant potential and enzyme
inhibition assay against AChE and BChE of anthranilic acid based sulfonamides has been
described. Sulfonamide derivative was firstly synthesized starting from methyl anthranilate,
which was further derivatized to give hydrazide based Schiff bases. These synthesized
compounds were then evaluated for antioxidant and enzyme inhibition potential against AChE
and BChE. The results showed that most of the compounds showed good enzyme inhibition
activity against AChE and BChE. The experimental findings were further supported by
molecular docking studies. The molecular docking results are coinciding with the experimental
results. These findings therefore encouraged to synthesize new hybrid compounds having Schiff
base and sulfonamide moieties together which can be optimized to be used as potent AChE and
BChE inhibitors.