Synthesis, characterization, biological evaluation and molecular docking studies of N-functionalized derivatives of 2-aminobenzohydrazide

Article abstract of DOI:10.1016/j.molstruc.2020.128042

In this paper, N-functionalized derivatives of methyl anthranilate (1–3) were prepared by reacting it with various acid chlorides to give amide derivatives (1–3) which were further converted to benzohydrazide derivatives (4–6) by reacting them with hydraz

Full text of DOI:10.1016/j.molstruc.2020.128042

Journal Pre-proof
Synthesis, characterization, biological evaluation and molecular docking studies of N-
functionalized derivatives of 2-aminobenzohydrazide
Naghmana Kausar, Shahzad Murtaza, Muhammad Nadeem Arshad, Robina Rashid,
Abdullah M. Asiri, Noman Javid, Mulazim Hussain Asim, Zaman Ashraf
PII:
S0022-2860(20)30367-7
DOI:
https://doi.org/10.1016/j.molstruc.2020.128042
MOLSTR 128042
Reference:
To appear in: Journal of Molecular Structure
Received Date: 27 January 2020
Revised Date: 6 March 2020
Accepted Date: 8 March 2020
Please cite this article as: N. Kausar, S. Murtaza, M.N. Arshad, R. Rashid, A.M. Asiri, N. Javid, M.H.
Asim, Z. Ashraf, Synthesis, characterization, biological evaluation and molecular docking studies of N-
functionalized derivatives of 2-aminobenzohydrazide, Journal of Molecular Structure (2020), doi: https://
doi.org/10.1016/j.molstruc.2020.128042.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. Please note that,
during the production process, errors may be discovered which could affect the content, and all legal
disclaimers that apply to the journal pertain.
© 2020 Published by Elsevier B.V.

Credit Author Statement
Naghmana Kausar: Methodology, Data curation, Writing- Original draft preparation, Software. Shahzad
Murtaza.: Supervision. Conceptualization, Writing- Reviewing and Editing, Muhammad Nadeem Arshad:
X-ray crystallographic analysis Robina Rashid: Free radical activity: Abdullah M. Asiri: X-ray
crystallographic analysis, Software, Validation: Noman Javid: Structural Analysis. Mulazim Hussain
Asim: Biological evaluation. Zaman Ashraf: Biological evaluation.

Synthesis, characterization, biological evaluation and molecular docking
studies of N-functionalized derivatives of 2-aminobenzohydrazide:
*
*
Naghmana Kausar a , Shahzad Murtaza a , Muhammad Nadeem Arshad b, c, Robina Rashid d,
Abdullah M. Asiri b, c, Noman Javid e, Mulazim Hussain Asim f, Zaman Ashraf g
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 Nawaz Sharif Medical College, University of Gujrat, 50700, Gujrat, Pakistan.
e Department of Chemistry, Forman Christian College, Lahore, Pakistan.
f Faculty of Pharmacy, Department of Pharmaceutics, University of Sargodha, 40100
Sargodha, Pakistan
gDepartment of Chemistry, Allama Iqbal Open University, Islamabad, Pakistan
Corresponding Author:
Dr. Shahzad Murtaza
Associate Professor, Department of Chemistry,
Faculty of Science, University of Gujrat, Gujrat, Pakistan
Email: shahzad.murtaza@uog.edu.pk

Abstract:
In this paper, N-functionalized derivatives of methyl anthranilate (1-3) were prepared by
reacting it with various acid chlorides to give amide derivatives (1-3) which were further
converted to benzohydrazide derivatives (4-6) by reacting them with hydrazine. These N-
functionalized derivatives of 2-aminobenzohydrazide (4-6) were reacted with 4-
triflouromethyl benzaldehyde to give schiff base derivatives (7-9). All the synthesized
compounds (1-9) were characterized by Mass spectrometry as well as NMR and FTIR
spectroscopic techniques. Single Crystal X-ray diffraction analysis technique (XRD) was
utilized to analyze crystalline compound 1. AChE and BChE enzymes inhibition potential
was checked for the synthesized compounds. Putative binding approaches of screened
compounds were explored by molecular docking studies. Synthesized compounds were also
checked for antioxidant activity. Enzyme inhibition analysis showed that compounds 2 and 9
exhibited good AChE inhibition showing 68% and 60% percentage inhibition, respectively,
while very good BChE inhibition activities were shown by compounds 2 (70%) and 5 (76%).
Enzyme inhibition results were also supported by molecular docking studies with lowest
binding energy values of -9.79 Kcal mol-1 for BChE and -9.55 Kcal mol-1 for AChE for
compound 9. Hydrazide derivatives (4-6) showed very significant results for antioxidant
activity with percentage scavenging greater than 90%.
Keywords: Benzohydrazide, heterocyclic, enzyme inhibition, crystal description,
sulfonamide, DPPH
Graphical Abstract:
1. Introduction
The development of new biologically important molecules is decisive to cater with diseases
and for improved health conditions. The incorporation of pharmacophoric moiety on food-

grade molecule could be a promising approach to get valuable molecules. Methyl 2-
aminobenzoate (methyl anthranilate) is used as a flavouring agent with fruity grape smell and
chemically consist of two important functional groups (amino and ester). In this research
work, these functional groups were derivatized to get amides, sulfonamides, benzohydrazides
and schiff bases. Benzohydrazides are the aromatic derivatives of hydrazine and benzoic acid
derivatives. Now a day, benzohydazide derivatives have become very important due to their
various pharmacological actions. They are found to act as analgesics [1] and anti-
inflammatory agents [2]. Benzohydrazides derivatives have also shown promising
antimicrobial activities [3] along with their importance as anti-cancerous drugs [4].
Significant anti-tubercular [5] and anti HIV activities [6] are being possessed by
benzohydrazide derivatives. They have also been found to possess significant AChE and
BChE inhibition activities [7-8] where flouro substituted derivative of benzohydrazides have
shown considerable AChE inhibition activities [9-10]. Because of possessing a large range of
biological activities, benzohydrazide moiety can be considered as an important
pharmacophore.
Sulfonamides have also attracted the attention of the researchers because of their diverse
pharmaceutical importance due to possessing anti-cancerous [11], HIV protease inhibition
[12], antibacterial [13] and anti-Alzheimer properties [14]. Schiff base derivatives possessing
versatile therapeutic activities such as anti-HIV and anti-cancerous activity [15], antitumor
activities [16] and antifungal activities [17] are also getting eminence. Amides have their own
biological significance due to showing various important biological activities such as N-
acylethanolamine acid amidase (NAAA) inhibitors [18], anticancer [19] and antimicrobial
agents [20].
Enormous biological importance of schiff bases, amides, sulfonamides and benzohydrazide
derivatives has urged us to design this research work to synthesize schiff base derivatives of
2-aminobenzohydrazide with functionalization of amino group with substituents of various
types including sulfonamide along with amides, of both aromatic as well as heterocyclic
nature and further investigated these compounds for their prospective of enzyme inhibition
against BChE as well as AChE enzymes and antioxidant potential.
2. Materials and Methods

All the chemicals including methyl anthranilate, hydrazine hydrate, 4-bromobezenesulfonyl
chloride, benzoyl chloride, thiophene-2-carbonyl chloride and aldehyde were bought from
Sigma-Aldrich (USA) and Merck (Germany) and were utilized as received. Electrothermal
melting point apparatus was utilized for measuring melting points and these melting points
are uncorrected. FTIR spectra were recorded on a Bio-red spectrophotometer using KBr
discs. NMR spectra were taken using JEOL- 300 MHz DELTA2_NMR Spectrometer (1H,
300 MHz and 13C, 75 MHz) utilizing DMSO-d6 as solvent. Mass spectra were taken by
Tandem Mass Spectrometric analysis (LTQ XL Linear Ion Trap Mass Spectrophotometer,
Thermo Scientific, USA), using Electrospray (ESI) ionization probe.
2.1. Synthesis of N-functionalized derivatives of methyl anthranilate (1-3)
Weighed quantity of methyl anthranilate (1.29 mL, 10.0 mmol) was taken and its solution
was prepared in pyridine (10 mL). Solutions of acid chlorides (15mmol) in distilled methanol
were added drop wise to the methyl anthranilate solution followed by stirring of reaction
mixture. pH of the solution was kept up to 7 by constantly adding sodium carbonate. The
products precipitated out from the reaction mixture, which were washed with cold, distilled
methanol. Obtained products were recrystallized using methanol to get pure products in
decent yield. (Scheme 1)
2.2. Synthesis of hydrazides of N-functionalized derivatives of methyl anthranilate (4-6)
Weighed amount of N-functionalized derivatives of methyl anthranilate (1-3) were dissolved
in acetonitrile. Excess hydrazine was added to the reaction mixture and was subjected to
refluxing for two hours. Excess solvents were removed under vacuum. Ethyl acetate /water
extraction was performed to remove excess hydrazine. Ethyl acetate was vaporized under
reduced pressure to get crude products. Recrystallization of the products was done using
ethanol to get pure products. (Scheme 1)
2.3. Synthesis of Schiff bases of hydrazides of N-functionalized derivatives of methyl
anthranilate (6-9)
Stoichiometric amounts of synthesized hydrazides (4-6) and 4-triflouromethyl benzaldehyde
were dissolved in methanol (10 mL). The reaction mixture was refluxed for two hours.
Solvents were vaporized under vacuum. Washing and recrystallization of the product was

done using cold ethanol and methanol, respectively to get products in decent yield (about
90%). (Scheme 1)
O
CH3
O
H2N
O
Pyridine
R-Cl
NH
+
Stirring, 2 Hrs
R
H3C
O
O
(1 - 3)
H2N NH2
CF3 OHC
O
NH2
NH
N
CF3
NH
Methanol
NH
R
NH
R
(7 - 9)
(4 - 6)
Where
O
O
S
O
S
O
Br
R =
(1, 4, 7), R =
(2, 5, 8), R =
(3, 6, 9)
Scheme 1.
2.4. Characterization
The physico-chemical data for the synthesized compounds is given below
Methyl 2-{[(4-bromophenyl)sulfonyl]amino}benzoate (1)
M.F: C14H12BrNO4S; Yield: 64%; m.p. 110±1°C; 1H-NMR (300 MHz, DMSO-d6): δ = 10.43
(1H, br, s, NH-SO2), 7.18-7.84 (8H, m, Aromatic CH), 3.81 (3H, s, CH3O). 13C NMR (75
MHz, DMSO-d6): δ = 167.7 (C=O), 138.5, 138.3, 134.7, 132.9 (2), 131.4, 129.3 (2), 127.8,
124.9, 121.3, 119.6 (2 benzene rings), 53.0 (CH3O). FTIR (cm−1): 3188 (NH, stretch), 1686
(ester C=O, stretch), 1342 (S=O, stretch), 1261 (C - O, stretch). MS (ESI): m/z (%), (369
(25), [M]-.), 311 (10), 220 (27), 214 (28).

Methyl 2-benzamidobenzoate (2)
1
M.F: C15H13NO3; Yield: 75%; m.p. 104±1°C; H-NMR (300 MHz, DMSO-d6): δ = 11.17
(1H, br, s, NHCO), 7.35-8.38 (9H, m, Aromatic CH), 3.47 (3H, s, CH3O). 13C NMR (75
MHz, DMSO-d6): δ = 167.6 (COO), 165.9 (CONH), 142.6, 138.4, 135.7 (2), 131.5 (2), 129.3,
126.8 (2), 124.7, 121.6, 120.5 (benzene rings), 52.6 (CH3O). FTIR (cm−1): 3263 (NH,
stretch), 3023 (C-H Stretch), 1691 (ester C=O, stretch), 1667 (C=O, stretch), 1267 (C-O,
stretch). MS (ESI): m/z (%), (254 (30), [M]-.), 224(12), 196 (24), 178 (27).
Methyl 2-(thiophene-2-carboxamido)benzoate (3)
1
M.F: C13H11NO3S; Yield: 78%; m.p. 131±1°C; H-NMR (300 MHz, DMSO-d6): δ = 11.37
(1H, br, s, NHCO), 7.32-8.21 (7H, m, Aromatic CH), 3.81 (3H, s, CH3O). 13C NMR (75
MHz, DMSO-d6): δ = 166.9 (COO), 160.8 (CONH), 143.2, 140.1, 135.2, 134.7, 131.6 (2),
129.8, 124.9, 121.2, 117.1 (aromatic rings), 54.1 (CH3O). FTIR (cm−1): 3262 (NH, stretch),
3087 (C-H Stretch), 1686 (ester C=O, stretch), 1665 (C=O, stretch), 1250 (C-O, stretch). MS
(ESI): m/z (%), 260 (20, [M]-.), 230(12), 202 (24), 178(26)
4-Bromo-N-(2-(hydrazinecarbonyl)phenyl)benzene sulfonamide (4)
1
M.F: C13H12BrN3O3S; Yield: 62%; m.p. 165±1°C; H-NMR (300 MHz, DMSO-d6): δ =
11.62 (1H, br, NH-NH2), 10.21 (1H, br, s, NH-SO2), 7.10-7.75 (8H, m, Aromatic CH), 4.84
(2H, br, NH2, amine). 13C NMR (75 MHz, DMSO-d6): δ = 163.7 (NHCO), 147.2, 144.7,
138.3, 135.6 (2), 132.7, 130.5 (2), 128.9, 125.6, 122.1, 119.2 (benzene rings). FTIR (cm−1):
3559 (NH2 anti-sym. stretch), 3385 (NH2 sym. stretch), 3331 (CON-H, Stretch), 3273 (SO2N-
H, stretch), 1630 (C=O, stretch), 1336 (S=O, stretch). MS (ESI): m/z (%), (370 (100), [M]-.),
354 (16), 310 (15), 221 (21), 200 (10), 171 (10).
N-(2-(hydrazinecarbonyl)phenyl)benzamide (5)
1
M.F: C14H13N3O2; Yield: 71%; m.p. 192±1°C; H-NMR (300 MHz, DMSO-d6): δ = 12.57
(1H, s, NH-CO), 10.21 (1H, br, NH-NH2), 7.17-8.68 (8H, m, Aromatic CH), 4.70 (2H, br,
NH2, amine). 13C NMR (75 MHz, DMSO-d6): δ = 167.9 (CONHNH2), 164.6 (NHCO), 139.7,
134.9, 132.5 (2), 129.4 (2), 128.1, 127.4 (2), 123.3, 120.6, 119.5 (benzene rings). FTIR
(cm−1): 3316 (NH2 stretch), 3204 (CON-H, Stretch), 3054 (C-H, stretch), 1646 (C=O,
stretch). MS (ESI): m/z (%), 254 (40, [M]-.), 240 (35), 236 (11), 196(10).

N-(2-(hydrazinecarbonyl)phenyl)thiophene-2-carboxamide (6)
1
M.F: C12H11N3O2S; Yield: 80%; m.p. 217±1°C; H-NMR (300 MHz, DMSO-d6): δ = 12.57
(1H, br, s, NH-CO), 10.22 (1H, br, NH-NH2), 7.14-8.54 (7H, m, Aromatic CH), 4.70 (2H, br,
NH2, amine). 13C NMR (75 MHz, DMSO-d6): δ = 167.9 (CONHNH2), 159.5 (NHCO), 140.2,
139.3, 132.8, 132.6, 128.7 (2), 128.1, 123.3, 120.5, 119.2 (aromatic rings). FTIR (cm−1):
3375 (NH2 anti-sym. stretch), 3314 (NH2 sym. stretch), 3213 (CON-H, Stretch), 3083 (C-H,
stretch), 1630 (C=O, stretch). MS (ESI): m/z (%), (260 (40), [M]-.), 246 (60), 229 (10), 202
(20), 176 (40), 137 (10).
(E)-4-bromo-N-(2-(2-(4-(trifluoromethyl)benzylidene)hydrazine-1-carbonyl)phenyl)benzene-
sulfonamide (7)
1
M.F: C21H15BrF3N3O3S; Yield: 91%; m.p. 195±1°C; H-NMR (300 MHz, DMSO-d6): δ =
12.03 (1H, br, s, NH-N), 10.59 (1H, br, s, NH-SO2), 8.42 (1H, s, N=CH), 7.26 – 7.97 (12H,
m, Aromatic CH). 13C NMR (75 MHz, DMSO-d6): δ = 164.6 (NHCO), 147.4 (CH=N), 138.4,
137.2, 133.0, 132.8 (3), 129.3 (3), 128.3 (2), 127.7, 126.2 (3), 125.1, 122.7 (2) (benzene
rings), 124.2 (CF3). FTIR (cm−1): 3326 (HN, stretch), 3089 (C-H, stretch), 1644 (C=O,
stretch), 1598 (N=CH, Stretch), 1322 (S=O, stretch). MS (ESI): m/z (%), (524 (20), [M]-.),
496 (70), 353 (55), 336 (05), 310 (20), 272 (100), 219 (05), 182(10).
(E) -N- (2-(2- (4-(trifluoromethyl)benzylidene)hydrazinecarbonyl)phenyl)benzamide (8)
M.F: C22H16F3N3O2; Yield: 91%; m.p. 229±1°C; 1H-NMR (300 MHz, DMSO-d6): δ = 12.12
(1H, br, s, NH-CO), 11.78 (1H, br, s, NH-N), 8.50(1H, s, N=CH), 7.38-8.59 (13H, m,
Aromatic CH). 13C NMR (75 MHz, DMSO-d6): δ = 165.2 (=N-NHCO), 164.9 (NHCO),
147.6 (CH=N), 138.7, 134.9, 134.2, 133.5, 132.7, 131.6, 130.2 (6), 129.7, 128.1 (2), 122.1,
121.6, 120.9 (benzene rings), 122.8 (CF3). FTIR (cm−1): 3192 (HN, stretch), 3067 (C-H,
stretch), 1657 (C=O, stretch), 1604 (N=CH, Stretch). MS (ESI): m/z (%), 410 (10, [M]-.), 239
(25), 221 (11), 187 (100), 159 (15).
(E)-N-(2-(2-(4-(trifluoromethyl)benzylidene)hydrazinecarbonyl)phenyl)thiophene-2-carbox-
amide (9)
M.F: C20H14F3N3O2S; Yield: 82%; m.p. 248±1°C; 1H-NMR (300 MHz, DMSO-d6): δ = 11.97
(1H, br, s, NH-CO), 11.91 (1H, br, s, NH-N), 8.51 (1H,s, N=CH), 7.21 – 8.45 (11H, m,
Aromatic CH). 13C NMR (75 MHz, DMSO-d6): δ = 165.6 (=N-NHCO), 163.2 (NHCO),
146.7 (CH=N), 141.3, 138.9, 138.1, 133.8, 132.5, 132.1, 130.9, 129.2 (2), 128.4, 127.3, 125.7

(2), 124.1, 122.9, 122.5 (aromatic rings), 126.4 (CF3). FTIR (cm−1): 3323 (HN, stretch),
3056 (C-H, stretch), 1640 (C=O, stretch), 1587 (N=CH, Stretch). MS (ESI): m/z (%), (416
(15), [M]-.), 388 (11), 332(12), 245 (06), 227 (10), 187 (100), 161 (15).
2.5. Crystallography
The selected sample of prismatic crystal was fixed, using glue, over the glass tip. This
assemblage was attached on the Agilent SuperNova (Dual source) Agilent Technologies
Diffractometer, furnished with microfocus Cu/Mo Kα radiation for the collection of data.
CrysAlisPro, software was utilized for the collection of data [21] maintaining temperature of
the system at 296 K taking Mo Kα radiation as source of light radiation. Direct method was
employed to solve structures utilizing SHELXS–97 [22] and refinement of the results was
performed by employing full–matrix least–squares methods on F2 utilizing SHELXL–97
[22]. Full–matrix least squares methods were utilized for the refinement of all non–hydrogen
atoms anisotropically [22]. PLATON [23] and ORTEP software packages were used for the
generation of figures [24].
There are three different types of hydrogen atoms available in the molecules i.e. aromatic,
methyl and amino hydrogen atoms. All types of hydrogen atoms were positioned
geometrically with the dC-H = 0.93 Å for hydrogen atoms of aromatic ring, dC-H =0.96Å for
carbon atom of methyl group and dN-H = 0.86Å. The Uiso(H) for nitrogen and aromatic
atoms (X = C & N) was set to 1.2 Ueq(X). For the methyl carbon atom, the Uiso(H) was
fixed to 1.5 Ueq(C). The crystal data was submitted to Cambridge Crystallographic Data
Centre, as CCDC No. 1976272, which can be obtained free of charges.
2.6. Biological Evaluation
2.6.1. Enzyme Inhibition Essay
Enzyme inhibition studies were performed by following well-known Ryan and Elman
technique [25] with little adjustment. Enzyme solutions (AChE/BChE) were mixed with
solutions of the synthesized compounds (1-9) followed by the addition of substrate solution
i.e., acetylthiocholine iodide for AChE and butyrylthiocholine chloride for BChE together
with DTNB solution and phosphate buffer of pH=8 and mixed properly. The mixture was
incubated for 30 minutes at 37 °C. Substrate hydrolysis was estimated by spectrophotometer
by recording absorbance at λ=400 nm for AChE and λ=412 nm for BChE. Enzyme inhibition
rate was measured by the following equation.

ꢊ − ꢋ
ꢊ
%ꢀꢁꢂ ꢃꢄℎꢅꢆꢅꢇꢅꢈꢄ = ꢉ
ꢌ × 100
Where X = Value of Absorption for enzyme including test sample
Y = Value of Absorption for enzyme devoid of test sample
Every test was performed thrice and mean was calculated. Donepzil was taken as reference.
2.6.2. Antioxidant analysis
DPPH method [26] was used for determining the antioxidant potential of the compounds (1-
9). Ethanolic solution of DPPH was mixed with the solution of the test compounds followed
o
by the incubation of the mixture at 37 C for 20 minutes. Extent of change in colour was
determined by spectrophotometry by recording the absorbance at λ=517 nm. For positive
control ascorbic acid was used. The equation operated to compute free radical scavenging
(%age) for each of test compound is given below
ꢐꢑ − ꢐꢒ
%ꢀꢁꢂ ꢍꢎꢀꢏꢂꢄꢁꢅꢄꢁ = ꢉ
ꢌ × 100
ꢐꢑ
Where
Ad = Absorbance of the DPPH solution,
As = Absorbance of the sample solution
2.7. Molecular Docking
For exploration of binding approaches of synthesized compounds with the enzymes, BChE
and AChE, molecular docking examination was executed. The proteins were taken from
Protein Data Bank. For the sketching of the molecular structures and 3D optimization,
ACD/ChemSketch and Chem 3D Pro 12.0, soft ware packages were utilized. The structures
were saved as SYBYL mol2 document form. The software used to carry out docking
procedure was AutoDock Tool v1.5.6. In this docking procedure, hundred diverse
conformations were obtained and was chosen superior amongst all the conformations in the
wake of imagining each posture by utilizing Discovery Studio Visualizer v4.0.23 [27].
3. Results and Discussion
3.1. Chemistry

N-functionalized derivatives of methyl anthranilate were synthesized according to Scheme 1
as reported in literature [28]. Methyl anthranilate has two active sites i.e., an amino group and
an ester linkage on which transformations can be done. Firstly its amino group was
functionalized by carrying out its reaction with different acid chlorides using pyridine to
obtain compounds (1-3). Then the ester linkage of these N-functionalized derivatives was
converted to their hydrazide derivatives (4-6) by reacting them with hydrazine hydrate. These
synthesized benzohydrazide derivatives (4-6) were further transformed to their schiff base
analogues by reacting them with 4-triflouromethylbenzaldehyde to synthesize compounds (7-
9). 1H-NMR, 13C NMR, FTIR and X-ray-Crystallographic techniques were employed to carry
out characterization of the synthesized compounds.
3.1.1. FT-IR spectral analysis
Different functional groups present in all the synthesized compounds were determined by
FTIR spectral analysis. Shifting of the signal of (C=O) group of ester linkage in compounds
(1-3) from ῡ = 1691 cm-1 – 1686 cm-1 to lower values of ῡ = 1646 cm-1 – 1630 cm-1 in
compounds (4-6) and also the appearance of signals for NH2 at ῡ = 3559 cm-1 – 3314 cm-1
confirmed the formation of hydrazide linkage. Similarly formation of imine linkage in
compounds (7-9) was authenticated with the appearance of at ῡ = 1604 cm-1 – 1588 cm-1 (N=
CH).
3.1.2. 1H-NMR spectral analysis
1H-NMR spectral data also confirmed the structures of all the synthesized compounds.
Signals of three protons of -OCH3 group of ester linkage in compounds (1-3) at δ = 3.47 ppm
– 3.81 ppm disappeared accompanying with the appearance of peaks at δ = 4.70 ppm – 4.84
ppm relating to –NH2 group in compounds (4-6) [28]. Singlet peaks were also observed for
one proton of NH-NH2 in the range of δ = 10.21 ppm – 11.62 ppm in these compounds (4-6).
Appearance of characteristic singlets between δ = 8.42 ppm – 8.51 ppm relating to the imine
linkage (N=CH) also gave confirmation of the conversion of compounds (4-6) to their schiff
base derivatives. Signals appeared in the range of 7.10 ppm – 8.68 ppm were due to aromatic
1
protons. H-NMR spectral data supported the structures of all the synthesized compounds
strongly.
3.1.3. 13C NMR spectral analysis

13C NMR spectral data also confirmed the structures of the synthesized compounds. Signals
appearing at of δ = 52.6 ppm – 54.1 ppm for –OCH3 carbon of ester linkage in compounds
(1-3) has disappeared in compounds (4-6) due to conversion of ester linkage into hydrazide in
these compounds. The signal for –HC=N- carbon atom of benzylidene group was appeared at
δ = 146.7 ppm – 147.6 ppm in compounds (7-9).
3.1.4. Mass spectroscopic analysis
Electrospray Ionization technique (ESI) was employed for the establishment of the structures
of the synthesized compounds. Characteristic peaks corresponding to [M-H] ions in -ve mode
appeared in mass spectrum of all compounds. In compound 5, cyclization leading to the
formation of quinazolinone nucleus gave peak at m/z = 236 [29].
3.1.5. Crystal Description
The title compound 2-(4-bromo-benzenesulfonylamino)-benzoic acid methyl ester (1)
belongs to the sulfonamide branch of organic chemistry. The molecule contains sulfonamide
and ester functional group in it as shown in Fig. 1 (a). The crystal data is being presented in
Table 1. The compound adopted V-shape where the dihedral angle between the two aromatic
rings (C1-C6) & (C7-C12) is 83.84(5)°. In the central sulfonamide functional group, the
geometry around the sulphur group is disordered tetrahedral geometry where the angles are
ranging from 105.47(2)° to 119.42(2)Å around it. The methyl acetate group
(O3/C13/O4/C14) is twisted by 1.77(3)° with reference to parent aromatic ring (C7-C12).
The molecules afford inter and intramolecular hydrogen bonding in their unit cell. A strong
intramolecular interaction like N1—H1N…O3 produce six membered ring motif with the
root mean square deviation value of about 0.1121 Å. This six membered ring motif is leaning
at dihedral angle of 77.39(2)° and 6.47(2) with reference to the aromatic rings (C1-C6) &
(C7-C12) respectively. A non-classical hydrogen bonding interaction where C3 is acting as
donor atom via H3 atom to the O1 oxygen atom by following the symmetry operation x+1, y,
z is depicted in Fig. 2(b). The hydrogen bond interactions are presented in Table 2. This
hydrogen bond association joins the molecules along a-axis to make long chain as presented
in Fig. 2 and supramolecular nature of compound. The bond angles and bond lengths are
shown in Table 3.

Fig. 1. a) Compound 1 ORTEP diagram, thermal ellipsoids sketched at 50% probability level.
b) Hydrogen bonding associations of both inter and intra-molecular type in a unit cell in
compound 1.
Fig. 2. Long chain formation along a-axis via C3-H3…O1 interaction
Table 1
Crystal data and structure refinement for compound 1
Crystal data and structure refinement for compound 1
CCDC number
Formula weight
Empirical formula
Crystal system
1976272
370.22
C14H12BrNO4S
monoclinic

Temperature/K
Space group
296(2)
P21/c
c/Å
b/Å
a/Å
12.2255(12)
15.6090(13)
8.0748(5)
γ/°
90
β/°
91.191(7)
α/°
Z
90
4
2.817
1540.6(2)
μ/mm-1
Volume/Å3
ρcalcg/cm3
1.596
0.43 × 0.28 × 0.15
744.0
Crystal size/mm3
F(000)
2Θ range for data collection/°
Radiation
5.682 to 58.462
MoKα (λ = 0.7107)
-11 ≤ h ≤ 10, -16 ≤ k ≤
21, -15 ≤ l ≤ 15
8864
Index ranges
Reflections collected
3662 [Rint = 0.0339,
Rsigma = 0.0446]
1.044
Independent reflections
Goodness-of-fit on F2
Data/restraints/parameters
3662/0/191
R1 = 0.1105, wR2 =
0.1835
Final R indexes [all data]
R1 = 0.0659, wR2 =
0.1554
Final R indexes [I>=2σ (I)]
Largest diff. peak/hole / e Å-3
0.92/-1.01
Table 2
Hydrogen bond associations in structure of compound 1.
D
H
A
d(D-H)/Å
0.93
d(H-A)/Å
2.36
2.02
11+X,+Y,+Z
d(D-A)/Å
3.278(5)
2.611(5)
D-H-A/°
171.0
124.9
C3 H3 O11
N1 H1N O3
0.86
Table 3
Selected Bond Angles and Bond lengths for Compound 1.
Bond Angles
Bond Lengths
Atom Atom Length/Å
C1 S1 1.763(4)
AtomAtomAtom
C6 C1 S1
Angle/˚
120.5(3)

C2 C1 S1
C3 C4 C5
C3 C4 Br1
C5 C4 Br1
C12 C7 N1
C8 C7 N1
O3 C13 O4
O3 C13 C8
O4 C13 C8
C7 N1 S1
C13 O4 C14
O1 S1 O2
O1 S1 N1
O2 S1 N1
O1 S1 C1
O2 S1 C1
N1 S1 C1
118.5(3)
122.2(5)
119.4(4)
118.4(4)
121.6(4)
118.8(3)
122.0(4)
124.6(4)
113.4(3)
127.2(3)
115.6(3)
119.42(18)
109.95(19)
104.52(18)
107.2(2)
109.42(18)
105.47(17)
C4 C5
C4 Br1
C5 C6
C7 C12
C7 N1
C13 O3
C13 O4
C14 O4
N1 S1
O1 S1
O2 S1
1.364(7)
1.895(5)
1.381(7)
1.390(6)
1.411(5)
1.207(5)
1.318(5)
1.445(5)
1.620(3)
1.422(3)
1.428(3)
3.2. Antioxidant activity
DPPH free radical scavenging method was used to find the antioxidant potential of the
synthesized compounds. Literature has discovered that the compounds having reducing
properties converted DPPH to 1, 1-diphenyl-2-picrylhydrazine [30]. DPPH having deep
violet color absorbed light at λ= 517 nm due to the delocalization of free electrons [31]. The
compounds owning antioxidant potential do pair up the single electron of DPPH, cause
reduction in the absorption at the λmax of DPPH. Ascorbic acid as an antioxidant was chosen
as reference. N-functionalized derivatives of methyl anthranilate (1-3) exhibited moderate
antioxidant activity whereas, hydrazide derivatives of N-functionalized methyl anthranilate
(4-6) showed very good antioxidant activity. Higher antioxidant activity could be due to the
presence of –NH2 group. The relatively easier availability of the hydrogen atoms of NH2
could make them as an efficient reducing agent [32, 33]. Schiff base derivatives (7-9) have
shown reduction in antioxidant activity. However by proper selection of the substituent on the
schiff bases can lead to their improved antioxidant potential. The results are described as
%age scavenging activity and are displayed in Table 4.
Table 4
Scavenging potential (%) of compounds 1-9 at 1μg/μl concentration

Percentage
scavenging
Compounds
Absorbance
1.14
1.27
1.33
0.083
0.080
0.081
1.46
43.87
37.46
34.51
93.91
95.06
92.01
28.11
29.59
27.62
1
2
3
4
5
6
7
8
9
1.43
1.47
+ve Control
(Ascorbic acid)
DPPH
0.08
96.06
2.031
3.3. Enzyme inhibition studies
Screening of the compounds (1–9) in vitro was done against acetyl cholinesterase and butyryl
cholinesterase enzymes for their inhibition by utilizing spectrophotometric technique taking
Donepzil as positive control. Results are described in terms of %age inhibition of enzyme
activities as presented in Table 5. Results clearly indicated that the compounds 2, 3 and 9
showed good inhibition potential against AChE whereas, 2, 4 and 5 against BChE. It is
significant that the methyl 2-benzamidobenzoate (2) was established to be the two fold
inhibitor of AChE as well as BChE which directed that it could be utilized as dual inhibitor
for both the enzymes and for treatment of over expression of both the enzymes. However,
compound methyl 2-(thiophene-2-carboxamido)benzoate (3) was also found to have
considerable activity against both BChE and AChE enzyme. In general, hydrazide derivatives
of N-functionalized methyl anthranilate (4-6) showed good inhibition against BChE as
compared to AChE. The conversion of the hydrazide derivatives (4-6) into schiff bases (7-9)
had overall reduced the inhibition potential against both the enzymes. In case of N-
derivatization of methyl anthranilate with thiophenyl carbonyl group caused the overall
increase in the inhibition activity against both the enzymes as depicted by inhibition activities
of compound 3, 6 and 9. These results of enzyme inhibition were further reinforced by
molecular docking studies. By following the results of our recent study, we planned to carry
on the synthesis of new N-functionalized derivatives of 2-aminobenzohydrazide
accompanying with little modifications on compound 9 by altering substituents to synthesize
a series of compounds which could act as an effective class of BChE and AChE inhibitors.

Table 5
AChE and BChE inhibition (%) of compounds 1-9 at concentration of 50μM.
Compound #
Percentage Inhibition
Percentage Inhibition
against AChE
against BChE
24
68
54
30
39
52
34
18
60
95
32
70
59
61
76
51
56
24
42
92
1
2
3
4
5
6
7
8
9
Donepzil
3.4. Molecular Docking Studies
Molecular docking studies were executed to discover probable binding interactions of
inhibitor compounds with amino acids of active site of the enzymes taken and also to certify
the experimental findings. Furthermore, to establish SAR studies, these expected
interpretations were utilized to discover interactions between protein and ligand at molecular
level. Templates taken were human AChE (PDB ID: 4BDT) and BChE (PDB ID: 4BDS) X-
ray structures to carry the process of molecular docking. The lowest bonding energies of the
compounds (1–9) found after docking investigation are presented in Table 6.
Table 6
Lowest binding energies related to selected conformations against human AChE and BChE
Compound
Lowest Binding Energy
Lowest Binding Energy
#
ΔG (kJ mol−1) h AChE
ΔG (kJ mol−1) h BChE
-9.27
-7.55
-7.51
-7.05
1
2
-7.51
-7.33
3
-9.07
-8.72
4
-7.43
-7.08
5
-7.96
-6.98
6
-10.46
-9.53
-9.55
-9.64
-9.50
-9.79
7
8
9
−10 (HUW)
−6.83 (THA)
Standard
The molecular docking studies revealed that a wide variety of interactions have been
established by most stable conformations of all the inhibitors with the amino acid residues of

the active sites of BChE and AChE and most of them were well accommodated there. The
most active compound 2, when visualized in the active site of human AChE established a lot
of significant binding interactions as represented in Fig. 3. This inhibitor utilized oxygen
atom of its ester linkage to make H-bonding interactions with AChE, catalytic triad amino
acid residue Ser-203 with distance 3.03 Å. Similarly, compound 2 made H-bonding
interaction using ester oxygen and another π-donor hydrogen bond association with electron
deficient hydrogen atom of –OH group attached to amino acid residue Tyr-337 of AChE
(3.18 Å). In the same way, carbonyl oxygen of amide linkage of compound 2 was involved in
H-bonding interactions with –OH group of TYR-124 (3.22 Å) and SER-125 (2.90 Å). Trp-86
established π-sigma binding with aromatic ring of the benzamide linkage with distance of
3.68 Å). This compound also utilized its aromatic ring in forming amide π-stacked bonding
with Gly-121 and Gly-122, amino acid residues of AChE (4.32 Å).
In the same way, many important interactions were revealed upon visualization of compound
2 in active site of BChE enzyme. It established two H-bonding interactions, one by utilizing
its NH hydrogen atom with Ser-198 (2.26 Å), amino acid residue from BChE catalytic triad
and second with amino acid residue of oxyanion hole, Ala-199 through its carbonyl oxygen
atom (2.55 Å). Compound 2 also established carbon hydrogen bond with Glu-197, amino
acid residue of BChE with distance of 3.78 Å. Trp-82, an amino acid residue from choline
binding site established π-sigma interactions with methyl group of ester linkage (3.85 Å).
Leu-286, amino acid from hydrophobic pocket of BChE made π-π stacked bonding with the
under observed compound with distance of 5.04 Å. The expected binding interactions of
compound 2 with BChE can be seen in Fig. 4.

Fig. 3. Putative binding approaches of compound 2 inside AChE enzyme.

Fig. 4. Compound 2 interactions with BChE at 3D space; Interactions of the compound
displayed in the box are with specific amino acid residues of the enzyme. Enzyme is
exemplified by 3D ribbon; stick model is the lowest energy conformation of the compound 2.
3.5. Conclusion
Schiff bases, amides and sulfonamides of benzohydrazide were synthesized in good yield. All
the synthesised molecules were characterised by spectroscopic techniques. Their potential as
antioxidant and as anticholinesterases was also evaluated. The benzoyl,
thiophenecarboxamide and sulfonamide benzohydrazides showed excellent antioxidant
activities. The amide benzohydrazides were emerged as the potential candidate as
anticholinesterases since having good inhibition potential against both BChE and AChE. This
study may be further extended with different amide bezohydrazide derivatives to find out
potential molecules against Alzheimer disease.
Acknowledgement
The authors are highly thankful to Dr. Mazhar Iqbal, National Institute for Biotechnology &
Genetic Engineering (NIBGE), Faisalabad, Pakistan for providing assistance in LC-MS
analysis of the compounds and Higher Education Commission (HEC), Pakistan, for financial
support for the said analysis.
References
1. A. Deep, S. Jain, P.C. Sharma, P. Phogat, M. Malhotra, Synthesis of 2-(aryl)-5-
(arylidene)-4-thiazolidinone derivatives with potential analgesic and anti-inflammatory
activity. Med. Chem. Res. 21(8) (2012) 1652-1659.
2. R. Jarapula, K. Gangarapu, S. Manda, S. Rekulapally, Synthesis, in vivo anti-
inflammatory activity, and molecular docking studies of new isatin derivatives. Int. J.
Med. Chem. (2016).
3. R. Manivannan, R. Shopna, Anti-microbial and Anti-inflammatory Activity of New 4-
methoxy-3-(methoxymethyl) Phenol and (E)-N′-(5-bromo-2-methoxybenzylidene)-4-
methoxy Benzohydrazide Isolated from Calotropis gigantean white. Nat. Prod. Sci. 23(1)
(2017) 69-74.
4. A. Katiyar, M. Hegde, S. Kumar, V. Gopalakrishnan, K.D. Bhatelia, K. Ananthaswamy,
S.C. Raghavan, Synthesis and evaluation of the biological activity of N′-[2-oxo-1, 2
dihydro-3 H-indol-3-ylidene] benzohydrazides as potential anticancer agents. RSC Adv.
5(56) (2015) 45492-45501.

5. V.B. Tatipamula, G.S. Vedula, Antimicrobial and anti-tubercular activities of isolates and
semi synthetic derivatives of lichen Ramalina leiodea (Nyl.) Nyl. J. Serb. Chem. Soc.
84(6) (2019) 555-562.
6. A. Balachandran, R. Wong, P. Stoilov, S. Pan, B. Blencowe, P. Cheung, A. Cochrane,
Identification of small molecule modulators of HIV-1 Tat and Rev protein accumulation.
Retrovirology, 14(1) (2017) 7.
7. N. Karaman, Y. Sıcak, T. Taşkın-Tok, M. Öztürk, A. Karaküçük-İyidoğan, M. Dikmen,
E.E. Oruç-Emre, New piperidine-hydrazone derivatives: Synthesis, biological evaluations
and molecular docking studies as AChE and BChE inhibitors. Eur. J. Med. Chem. 124
(2016) 270-283.
8. M.U. Nisa, M.A. Munawar, A. Iqbal, A. Ahmed, M. Ashraf, A.G. Qurra-tul-Ann, M.A.
Khan, Synthesis of novel 5-(aroylhydrazinocarbonyl) escitalopram as cholinesterase
inhibitors. Eur. J. Med. Chem. 138 (2017) 396-406.
9. A.B.
Özçelik,
M.
Gökçe,
I.
Orhan,
M.F.
Inhibitory
Sahin,
Synthesis
Activities
and
of
Acetylcholinesterase/Butyrylcholinesterase
(Substituted/Nonsubstituted Benzal)Hydrazone Derivatives of 3-(6-Substituted-3(2H)-
Pyridazinon-2-yl)propionohydrazides. FABAD J. Pharm. Sci. 35(3) (2010) 153-161.
10. B. Kaya, Y. Özkay, H.E. Temel, Z.A. Kaplancıklı, Synthesis and biological evaluation of
novel
piperazine
containing
hydrazone
derivatives.
J.
Chem.
(2016)
DOI:10.1155/2016/5878410.
11. J. Mun, A.A. Jabbar, N.S. Devi, S. Yin, Y. Wang, C. Tan, D. Culver, J.P. Snyder, E.G.
Van Meir, M.M. Goodman, Design and in-vitro activities of N-alkyl-N-[(8-R-2,2-
dimethyl-2H-chromen-6-yl)methyl]heteroarylsulfonamides,
novel,
small-molecule
hypoxia inducible factor-1 pathway inhibitors and anticancer agents, J. Med. Chem. 15
(2012) 6738-6750.
12. E.D. Clercq, New developments in anti-HIV chemotherapy, Curr. Med. Chem. 8 (2001)
1543-1572.
13. B. Ahmed, S.A. Khan, T. Alam, Synthesis and antihepatotoxic activity of some
heterocyclic compounds containing the 1, 4-dioxane ring system, Pharmazie. 58 (2003)
173-176.
14. M. Markowicz-Piasecka, K.M. Huttunen, J. Sikora, Metformin and its sulphonamide
derivative simultaneously potentiateanti-cholinesterase activity of donepezil and inhibit
beta-amyloid aggregation, J. Enzyme Inhib. Med. Chem. 33 (1) (2018) 1309-1322.
15. D. Marton, U. Russo, D. Stivanello, G. Tagliavini, P. Ganis, G.C. Valle, Preparation of
Benzylstannanes by Zinc-Mediated Coupling of Benzyl Bromides with Organotin
Derivatives. Physicochemical Characterization and Crystal Structures, Organometallics.
15 (6) (1996) 1645–1650.
16. V.S.V. Satyanarayana, P. Sreevani, A.S. Kumar, V. Vijayakukar, Synthesis and
antimicrobial activity of new Schiff bases containing coumarin moiety and their spectral
characterization, ARKIVOC. 17 (2008) 221-233.
17. M. Yildiz, A. Kĺraz, B. Dülger, Synthesis and antimicrobial activity of new crown ethers
of Schiff base type, J. Serb. Chem. Soc. 72(3) (2007) 215-224.
18. P. Zhou, L. Xiang, D. Zhao, J. Ren, Y. Qiu, Y. Li, Synthesis, biological evaluation, and
structure activity relationship (SAR) study of pyrrolidine amide derivatives as N-
acylethanolamine acid amidase (NAAA) inhibitors. MedChemComm, 10(2) (2019) 252-
262.
19. V. Ramesh, G.P.C. Rao, D. Ramachandran, A.K. Chakravarthy, Synthesis and Biological
Evaluation of Amide Derivatives of Imidazopyridine as Anticancer Agents. Russ. J. Gen.
Chem. 89(7) (2019) 1491-1495.

20. J.N. Soni, S. S. Soman, Synthesis and antimicrobial evaluation of amide derivatives of
benzodifuran-2-carboxylic acid. Eur. J. Med. Chem. 75 (2014) 77-81.
21. Agilent CrysAlis PRO Agilent Technologies, Yarnton, England (2012).
22. G.M. Sheldrick, A short history of SHELX, Acta Cryst. A. 64 (1) (2008) 112-122.
23. A.L. Spek PLATON-a Multipurpose Crystallographic Tool Utrecht University, Utrecht,
(2005).
24. L.J. Farrugia, WinGX and ORTEP for Windows: an update, J. Appl. Cryst. 45 (4) (2012)
849-854.
25. G.L. Ellman, K.D. Courtney, J.V. Andres R.M. Featherstone, A new and rapid
colorimetric determination of acetylcholinesterase activity. Biochem. Pharmacol. 7(2)
(1961) 88-95.
26. S. Murtaza, S. Shamim, N. Kousar, M.N. Tahir, M. Sirajuddin, U.A. Rana, Synthesis,
biological investigation, calf thymus DNA binding and docking studies of the sulfonyl
hydrazides and their derivatives. . J. Mol. Struct. 1107 (2016) 99-108.
27. K. Iftikhar, S. Murtaza, N. Kousar, A. Abbas, M.N. Tahir, Aminobenzoic acid derivatives
as antioxidants and cholinesterase inhibitors; synthesis, biological evaluation and
molecular docking studies, Acta. Pol. Pharm. 75(2) (2018) 385-396.
28. N. Kausar, S. Muratza, M.A. Raza, H. Rafique, M.N. Arshad, A.A. Altaf, S. Shafqat,
Sulfonamide hybrid schiff bases of anthranilic acid: Synthesis, characterization and their
biological potential. J. Mol. Struct.1185 (2019) 8-20.
29. W.S. El-serwy, N.A. Mohamed, E.M. Kassem, K. Mahmoud, M.M. Mounier, Synthesis,
biological evaluation and docking analysis of some novel quinazoline derivatives as
antitumor agents. Iran. J. Pharm. Res. 15(1) (2016) 179.
30. Z. Akar, M. Küçük, H. Doğan, A new colorimetric DPPH• scavenging activity method
with no need for a spectrophotometer applied on synthetic and natural antioxidants and
medicinal herbs. J. Enzyme inhib. Med. Chem. 32(1) (2017) 640-647.
31. S.B. Kedare, R.P. Singh, Genesis and development of DPPH method of antioxidant assay.
J. Food Sci. technol. 48(4) (2011) 412-422.
32. D. Amic, D. Davidovic-Amic, D. Beslo, V. Rastija, B. Lucic, N. Trinajstic, SAR and
QSAR of the antioxidant activity of flavonoids, Curr. Med. Chem. 14 (7) (2007) 827-845.
33. E. Khan, Z. Gul, A. Shahzad, M.S. Jan, F. Ullah, M.N. Tahir, A. Noor, Coordination
compounds of 4, 5, 6, 7-tetrahydro-1 H-indazole with Cu (II), Co (II) and Ag (I):
structural, antimicrobial, antioxidant and enzyme inhibition studies. J. Coord. Chem.
70(24) (2017) 4054-4069.

•
•
N-functionalized derivatives of 2-aminobenzohydrazide were synthesized.
The benzohydrazides bearing hydrogen donating groups showed good
Antioxidant activities.
•
•
Good enzyme inhibition activity against AChE and BChE were shown by
amide derivatives of benzohydrazides.
Molecular docking evaluation also supported the experimental results for
enzyme inhibition.

Declaration of Interest
The authors declare that there is no conflict of interest.