Synthesis and in vitro acetylcholinesterase and butyrylcholinesterase inhibitory potential of hydrazide based Schiff bases

Article abstract of DOI:10.1016/j.bioorg.2016.07.005

To discover multifunctional agents for the treatment of Alzheimer's disease, a series of hydrazide based Schiff bases were designed and synthesized based on multitarget-directed strategy. We have synthesized twenty-eight analogs of hydrazide based Schiff bases, characterized by various spectroscopic techniques and evaluated in vitro for acetylcholinesterase and butyrylcholinesterase inhibition. All compounds showed varied degree of acetylcholinesterase and butyrylcholinesterase inhibition when compared with standard Eserine. Among the series, compounds 10, 3 and 24 having IC₅₀ values 4.12?±?0.01, 8.12?±?0.01 and 8.41?±?0.06?μM respectively showed potent acetylcholinesterase inhibition when compared with Eserine (IC₅₀?=?0.85?±?0.0001?μM). Three compounds 13, 24 and 3 having IC₅₀ values 6.51?±?0.01, 9.22?±?0.07 and 37.82?±?0.14?μM respectively showed potent butyrylcholinesterase inhibition by comparing with eserine (IC₅₀?=?0.04?±?0.0001?μM). The remaining compounds also exhibited moderate to weak inhibitory potential. Structure activity relationship has been established. Through molecular docking studies the binding interaction was confirmed.

Full text of DOI:10.1016/j.bioorg.2016.07.005

Accepted Manuscript
Synthesis and in vitro Acetylcholinesterase and Butyrylcholinesterase Inhibi-
tory Potential of Hydrazide based Schiff Bases
Fazal Rahim, Hayat Ullah, Muhammad Taha, Abdul Wadood, Muhammad
Tariq Javed, Wajid Rehman, Mohsan Nawaz, Muhammad Ashraf, Muhammad
Ali, Muhammad Sajid, Farman Ali, Muhammad Naseem Khan, Khalid
Mohammed Khan
PII:
S0045-2068(16)30112-2
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.07.005
YBIOO 1925
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
3 April 2016
29 June 2016
6 July 2016
Please cite this article as: F. Rahim, H. Ullah, M. Taha, A. Wadood, M.T. Javed, W. Rehman, M. Nawaz, M. Ashraf,
M. Ali, M. Sajid, F. Ali, M.N. Khan, K.M. Khan, Synthesis and in vitro Acetylcholinesterase and
Butyrylcholinesterase Inhibitory Potential of Hydrazide based Schiff Bases, Bioorganic Chemistry (2016), doi:
http://dx.doi.org/10.1016/j.bioorg.2016.07.005
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Synthesis and in vitro Acetylcholinesterase and Butyrylcholinesterase Inhibitory Potential
of Hydrazide based Schiff Bases
a∗
a
b,c
d
a
Fazal Rahim, Hayat Ullah, Muhammad Taha, Abdul Wadood, Muhammad Tariq Javed, Wajid
a
a
e
f
g
Rehman , Mohsan Nawaz, Muhammad Ashraf , Muhammad Ali , Muhammad Sajid , Farman Ali,
a
h
Muhammad Naseem Khan, Khalid Mohammed Khan
Depatment of Chemistry, Hazara University, Mansehra-21300, Pakistan. Atta-ur-Rahman Institute for
Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar
Puncak Alam, Selangor, Malaysia, Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor,
Malaysia. Department of Biohemistry, Abdul Wali Khan University Mardan, Mardan-23200, Pakistan.
Department of Biochemistry and Biotechnology, The Islamia University of Bahawalpur, Bahawalpur
3100. Pakistan. Center for Advanced Drug Research, COMSATS Institute of Information Technology,
University Road, Abbottabad 22060, KPK, Pakistan. Department of Biochemistry, Hazara University,
Mansehra-21300, Pakistan.. H. E. J. Research Institute of Chemistry, International Center for Chemical
a
b
c
d
e
f
6
g
h
and Biological Sciences, University of Karachi, Karachi-75270, Pakistan.
Abstract
To discover multifunctional agents for the treatment of Alzheimer’s disease, a series of
hydrazide based Schiff bases were designed and synthesized based on multitarget-directed
strategy. We have synthesized twenty eight analogs of hydrazide based Schiff bases,
characterized by various spectroscopic techniques and evaluated in vitro for acetylcholinesterase
and butyrylcholinesterase inhibition. All compounds showed varied degree of
acetylcholinesterase and butyrylcholinesterase inhibition when compared with standard Eserine.
Among the series, compounds 10, 3 and 24 having IC50 values 4.12±0.01, 8.12±0.01 and
8
.41±0.06 µM respectively showed potent acetylcholinesterase inhibition when compared with
Eserine (IC50 = 0.85±0.0001 µM). Three compounds 13, 24 and 3 having IC50 values 6.51±0.01,
.22±0.07 and 37.82±0.14 µM respectively showed potent butyrylcholinesterase inhibition by
9
comparing with eserine (IC50 = 0.04±0.0001 µM). The remaining compounds also exhibited
moderate to weak inhibitory potential. Structure activity relationship has been established.
Through molecular docking studies the binding interaction was confirmed.
Keywords: Synthesis, Acetylcholinesterase, Butyrylcholinesterase, Molecular docking, Schiff
Bases, SAR
*
Corresponding Author: fazalstar@gmail.com, Tel.: 0092-335-9528343;
1

1
. Introduction
Alzheimer’s disease (AD) is a chronic and progressive neurodegenerative disorder that is
characterized by several symptoms including memory loss, disorientation, cognitive dysfunction
and behavioral disturbances [1-3]. AD is associated with deficit of cholinergic functions in the
basal forebrain and cortex [4, 5]. Since lack of cholinergic neurotransmission is considered to
play a critical role in the learning and memory diminishment of AD patients, it has been reported
that activating the cholinergic function by inhibiting cholinesterases can be a clinically effective
method for treatment of AD [6]. Cholinesterases (ChEs) such as acetylcholinesterase (AChE)
and butyrylcholinesterase (BChE) are key enzymes that play important roles in cholinergic
transmission by hydrolyzing the neurotransmitter acetylcholine [7]. AChE, which consists of
multiple subunits, is a membrane-bound enzyme and present in the brain, muscles and
cholinergic neurons. In the mammalian brain, most of the AChE exists in the membrane-bound
G4 form, but its level declines as the neurons degenerate [8]. It plays an important role in the
regulation of several physiological reactions by hydrolyzing the neurotransmitter acetylcholine in
cholinergic synapses [9, 10]. BChE is expressed in neuroglia and found in the intestine, liver,
kidney, heart, lung and serum. It plays a major role in the metabolism of ester containing
compounds [11]. It also can hydrolyze acetylcholine, and its levels do not decline, or may even
increase in AD [12]. Normally in the brain AChE is predominant but BChE activity rises while
AChE activity remains unchanged or diminishes in the brain of patients with AD [13]. Therefore,
a drug inhibiting both AChE and BChE may be preferable to selective AChE or BChE inhibitors.
During the past few years, synthetic ChEs inhibitors, including tacrine, donepezil, rivastigmine
and galanthamine, have been used for clinical treatment of AD. However, the use of these drugs
is limited because of insufficient activity and side-effects such as hepatotoxicity and
gastrointestinal disturbance [14-16]. Due to the adverse side-effects of synthetic ChEs inhibitors,
the development of non-toxic ChEs inhibitors as alternatives to synthetic drug is of great interest
among researchers, and many natural AChE inhibitors have been isolated from natural
bioresources [17-19].
Schiff bases represent an important group of organic compounds having various biological
activities such as urease, α-glucosidase, antiglycation and beta-glucuronidase [20-29] The study
of novel biologically significant Schiff bases has been drawing attention of chemists and
pharmacists [30]. Schiff bases from acyl-hyrazide having a wide range of biological actions are
2

also reported i.e. Adriamycin immune conjugates, antiparasitic activity by inhibiting proteinase
against Trypanosoma brucei, which causes sleeping sickness in humans, insecticidal, antimyco-
bacterial and antileishmanial [31-35] and different analogs of N-cyanoethyl hydrazide also
illustrated activity against β-glucuronidase enzyme [36].
Here we are reporting synthesis of some new derivatives of hydrazide based Schiff bases and
their acetylcholinesterase and butyrylcholinesterase inhibitory potential and molecular docking
studies.
2.0. Result and Discussion
2.1. Chemistry
The synthesis of hydrazide based Schiff bases were carried out in two steps. First methyl
salicylate (1mmol) was mixed with hydrazine hydrate (1mmol) in methanol (10 ml) as solvent
with 2-3 drops of acetic acid under continuous stirring for 4 hours to obtain hydrazide. The
reaction completion was monitored by TLC through depletion of starting material spot and
formation of hydrazide fluorescent spot established the reaction completion. After completion
distilled water was added into the reaction mixture which gives white precipitate. The product
obtained was then further purified by washing with n-hexane.
In step-2, the hydrazide was mixed with different aldehydes (1:1) in EtOH with 1-2 drops of HCl
and stirred for 2-3 hours to obtain various Schiff bases of hydrazones (1-28). The reaction
completion was checked by TLC. The Schiff bases obtained were then washed with ethanol and
n-hexane (Scheme-1).
Insert Scheme Here
Insert Table-1 Here
2
2
.2. Pharmacology
.2.1. In vitro Acetylcholinesterase activity
In the continuous of our effort: on enzyme inhibition [37-44]. We have synthesized twenty eight
analogs of hydrazide based Schiff bases which have varied degree of acetylcholinesterase
inhibition ranging in between 4.12±0.005 to 461.3±0.17 μM when compared with standard
3

eserine having IC50 values 0.85±0.0001. Among the series analogs 10, 3 and 24 having IC50
values 4.12±0.005, 8.12±0.006 and 8.41±0.06 µM showed good acetylcholinesterase inhibition.
Compound 3 having 1-(benzyloxy)-4-methylbenzene, compound 24 having 2,4-dichloro-6-
methylphenol while compound 10 having 9-methylanthracene.
Structure-activity relationship studies suggested that the acetylcholinesterase inhibitory activities
of this class of compounds are mainly dependent upon the substitutions pattern on the phenyl
ring. By comparing analog 21 having IC value 311.53±0.27 μM with analogs 25 and 26 having
5
0
IC50 values 311.42±0.21 and 369.15±0.25 μM respectively, it was observed all these analogs
have same substituents like bromo and fluoro group on phenyl part but position of substituents
are different. In analog 21 the bromo group is present at ortho position and fluoro group is
present at meta position, in analog 25 the bromo group is present at meta position and fluoro
group is present at para position, while in analog 26 the bromo group is present at para position
and fluoro group is present at ortho position. The little bit difference in the activity of these three
analogs may be due to the difference position of the substituents on the phenyl part.
If we compare analog 3 having IC50 value 8.12±0.01 μM with analog 16 having IC50 value
1
8.31±0.02 μM, we found that analog 3 is many fold better than analog 16. Analog 3 have
benzoyloxy group, while analog 16 have benzoyloxy and methoxy groups but the arrangement of
benzoyloxy group is different in them which confirm that the difference in position of
substituents greatly affect the inhibitory potentials of the analogs. Similar effect was also
observed in monohydroxy analogs such as analog 27 and analog 28. In this study, we observed
that either EWG or EDG on aldehydic phenyl part showed potential but the slight difference in
potential was mainly affected by the position of the substituent as well as in some cases the
number of substituent also play a role.
2.2.2. In Vitro butyrylcholinesterase activity:
The synthesized hydrazide based Schiff bases were also evaluated against butyrylcholinesterase
inhibitory potential and all the analogs showed variable degree of inhibition ranging in between
6.51±0.01 to 451.6±0.21 μM when compared with standard eserine having IC50 values
0.04±0.0001 [Table-2]. Among the series analogs 13, 24 and 3 having IC50 values 6.51±0.006,
9.22±0.07 and 37.82±0.14 µM respectively showed good butyrylcholinesterase inhibition.
Analogs 3 and 24 also showed good inhibition in case of acetylcholinesterase inhibitory
4

potential. The structure activity relationship in this case was almost similar to that of
acetylcholinesterase. To understand the binding interaction of the most active analogs molecular
docking study was performed.
Insert Table-2 Here
2
.3. Molecular docking:
All the 28 synthesized compounds were docked into the binding pocket of the enzymes AChE
acetylcholinesterase) and BChE (butyrylcholinesterase) separately in order to find the binding
(
interactions of the compounds with the protein. For docking studies the default parameters of the
MOE were used i-e Placement: Triangle Matcher, Rescoring 1: London dG, Refinement: Force
field, Rescoring 2: London dG for each ligand 10 conformations were made and saved. In all the
docked compounds, the top ranked conformations were saved in a separate database.
2
.4. Discussion:
The Schiff bases of hydrazide analogs were synthesized and their inhibitory activities were found
out experimentally. The highly potent compounds from the series were selected on the basis of
IC50, which was determined experimentally. Compound 10, 3 and 24 have best inhibitory
activities i-e with IC50 values 4.12±0.01, 8.12±0.01 and 8.41±0.06 respectively against AChE
and compound 1 has no inhibitory activity against AChE enzyme. The docking studies of the
compounds as well as the experimental activities of the compounds correlated well.
Compound 10 was found with a best fit in the binding pocket of the AChE with the docking
score -11.4705 and have shown good interactions with the active site residues of the receptor
protein Tyr 121 and Phe 330. Tyr121 formed hydrogen bonds with the hydrogen atoms of the
–NH and –OH moiety of the compound. Phe 330 was observed making an arene-arene
interaction with the π system of the anthracene moiety of the compound. The 3D interaction of
the compound with the receptor AChE protein was shown in the figure 1. The existence of a
polar (NH & OH) and non-polar end (anthracene) in this compound might be a reason of its high
enzymatic potency through which it interacts with both polar and non-polar residues of the
binding pocket of protein.
5

Insert Figures- 1 Here
Compound 3 showed good docking score of -12.1658 and formed four interactions with the
residues Tyr 121, Ser 122, Trp 279 and Tyr 334 as shown in the figure 2. Tyr 121 and Ser 122
formed hydrogen bonds with the –OH group of the phenolic moiety of the compound. Trp 279
made an arene-arene interaction with one of the benzene ring and a similar interaction was found
for Tyr 334 with another benzene ring of the (benzyloxy) benzene of the compound. Compound
3
and 10 both have structures of almost similar natures and hence same behavior in the binding
pocket.
Insert Figures- 2 Here
Compound 24 have good IC50 (8.41±0.06) as well as good docking score i-e -11.1093. This
compound had shown four interactions with the Asp 72, Tyr 121, Ser 122 and Tyr 334 as shown
in the figure 3. Tyr 121 and Ser 122 interact with the hydroxyl group of di-chloro phenol moiety
of the ligand at one end and Asp 72 with the OH of the phenolic group at the other end. Tyr334,
additionally, made an arene-arene linkage with the π electronic cloud of the same phenolic group
of the compound. The docking study revealed that the compounds having proper polarizability
and exposed delocalized electronic system in their structures may reflect good enzymatic
potential as shown in the above mentioned compounds. On the other hand a compound with low
polarizability, strong steric hindrance and non-availability of delocalized electrons may leads to
very poor or no inhibitory potential.
Insert Figures- 3 Here
For example Non-active compound 1 has shown no interactions having two bulky methoxy
groups with restricted electronic nature and strong steric hindrance. The orientation mode of this
compound in the binding of ACHE is shown in the figure 4.
Insert Figures- 4 Here
Compound 13, 24 and 3 were found to be more active against BChE among the series of the
compounds while compound 1 was found to be non-active against BChE. The IC50 values of
these potent and novel compounds were experimentally determined to be 6.51±0.01, 9.22±0.07
6

and 37.82±0.14 of compound 13, 24 and 3 respectively. The selected compounds of being highly
active, novel and potent were also supported by the docking study as these ligands have best
interactions as well as good binding scores with the respective protein BCHE. Compound 13, 24,
3
and 1 showed docking score values of -10.3662, -10.0558, -10.0230 and -9.1271 respectively
and formed good interactions with the BChE protein’s active site pocket. Compound 13 was
theoretically observed making seven interactions with the active residues of the pocket of the
BCHE protein. Gly115, Gly116 and Tyr128 act as hydrogen donors and involved in hydrogen
bonding with the carbonyl oxygen atom of the formamide of the compound 13. Tyr128 is
forming two additional bonds with the hydrogen atom of the hydroxyl moiety of the phenol and
hydrogen (H) atom of the –NH moiety of the formamide. Tyr 128 also interacts with the nitrogen
atom of the methanimine of the inhibitor. Thr 122 formed ion-induced interaction with the
oxygen atom of the –OH moiety of the phenol group of the ligand. The 3D binding interactions
of the compound with the receptor protein were shown in the figure 5.
Insert Figures- 5 Here
Compound 24 was bound to the BCHE protein in an adequate manner and made four interactions
in which one is arene-cation interaction; two are hydrogen bonding interactions and a
hydrophobic interaction. Tyr 332 is bound to the carbonyl oxygen of the acetamide via hydrogen
bonding. His 438 formed an arene-cation linkage with the benzene ring of the 2,4-dichloro-6-
methylphenol of the inhibitor. Asp 70 and Ser 79 formed interactions with the hydrogen atom of
the –OH moiety of the phenol group of the ligand. The 3D binding mode of the ligand with
residues was as shown in the figure 6.
Insert Figures- 6 Here
Compound 3 was involved in making two interactions with the Trp 82 and Tyr 128. An arene-
arene interaction was found in the Trp 82 and benzene ring of the phenol moiety of the ligand
sharing the π – π electrons. Tyr 128 was directly connected with the H-atom of the hydroxyl
moiety of the phenol and act as hydrogen acceptor and formed hydrogen bond. The 3D
interactions image of the residues with the compound was as shown in the figure 7.
Insert Figures- 7 Here
7

It was observed that the attachment of an etherial substituent (di-phenyl ether) leads to the poor
interactions mode of this compound. The behavior of compound 1 may strengthen this
supposition as the number of etherial groups increases in this compound.ψ
Compound 1 has shown no interactions with the residues of the BChE protein. The 3D binding
mode of compound 1 with the protein is as given in the figure 8.
Insert Figures- 8 Here
3
.0. Conclusion
In conclusion we have synthesized twenty eight analogs of hydrazide based Schiff bases and
evaluated for acetylcholinesterase and butyrylcholinesterase inhibition. All compounds showed
varied degree of acetylcholinesterase and butyrylcholinesterase inhibition when compared with
standard eserine. Among the series, compounds 10, 3 and 24 having IC50 values 4.12±0.01,
8
.12±0.01 and 8.41±0.06 µM respectively showed good acetylcholinesterase inhibition. Three
compounds 13, 24 and 3 having IC50 values 6.51±0.01, 9.22±0.07 and 37.82±0.14 µM
respectively also showed potent butyrylcholinesterase inhibition. The remaining compounds also
exhibited moderate to weak inhibitory potential.
4. Experimental Section
4.1. General Methods
The 1H-NMR spectra were performed in DMSO-d6 on Avance Bruker AM-300 and AMX-400
MHz NMR instruments. The chemical shifts (d) are given in ppm. TMS is used as external
reference. EI-MS were recorded on a Finnigan MAT 311A mass spectrometer with a MASPEC
Data system. Molecular docking was done to obtain the binding mode of the synthesized
compounds in the active site of the target enzymes. MOE (Molecular Operating Environment)
software was used for the molecular docking studies. All the docking calculations were carried
out on Intel (R) xenon (R) CPU E5620@2.40GHz system having 3.8GB RAM with the open
1
1.4 (X 86_64) operating platform.
8

4
.2. Preparation of the ligands:
The three dimensional structures of all the twenty eight synthesized compounds were generated
by using the builder tool of the MOE software [45, 46]. The synthesized compounds were 3D
protonated and then energy minimization of each compound was done by using the default
parameters of the MOE (gradient: 0.05, Force Field: MMFF94X). All the compounds were then
saved in mdb file for further evaluation in molecular docking.
4.3. Preparation of the proteins:
The 3D structures of the two target enzymes (ACHE and BCHE) were downloaded from the
protein databank (PDB) PDB id: 1ACL and 1POP respectively. Each protein was opened in
MOE, water molecules were removed from the proteins. Then 3D protonation of each enzyme
was carried out and after 3D protonation energy minimization was done for the stability of the
proteins by using the default parameters of the MOE.
4
.4. Acetylcholinesterase Assay
The AChE inhibition activity was performed according to the method [39] with slight
modifications. Total volume of the reaction mixture was 100 µL. It contained 60 µL Na H PO
2
4
buffer with concentration of 50 mM and pH 7.7. 10 µL test compound (0.5 mM well-1) was
added, followed by the addition of 10 µL (0.005 unit well-1) enzyme. The contents were mixed
and pre-read at 405 nm. Then contents were pre-incubated for 10 min at 37ºC. The reaction was
initiated by the addition of 10 µL of 0.5 mM well-1 substrate (acetylthiocholine iodide),
followed by the addition of 10 µL DTNB (0.5 mM well-1). After 15 min of incubation at 37ºC
absorbance was measured at 405 nm using 96-well plate reader Synergy HT, Biotek, USA. All
experiments were carried out with their respective controls in triplicate. Eserine (0.5 mM well-1)
was used as a positive control. The percent inhibition was calculated by the help of following
equation.
Inhibition (%) = Control – Test x 100
Control
IC50 values were calculated using EZ–Fit Enzyme kinetics software (Perrella Scientific Inc.
Amherst, USA).
9

4
.5. Butyrylcholinesterase Assay
The BChE inhibition activity was performed according to the method [47] with slight
modifications. Total volume of the reaction mixture was 100 µL containing 60 µL, Na2H PO4
buffer, 50 mM and pH 7.7. Ten µL test compound 0.5 mM well-1 was added followed by the
addition of 10 µL (0.5 unit well-1) BChE (Sigma Inc.). The contents were mixed and pre-read at
4
1
05 nm and then pre-incubated for 10 min at 37ºC. The reaction was initiated by the addition of
0 µL of 0.5 mM well-1 substrate (butyrylthiocholine chloride). Followed by the addition of 10
µL DTNB, 0.5 mM well-1. After 15 min of incubation at 37ºC, absorbance was measured at 405
nm using 96-well plate reader Synergy HT, Biotek, USA. All experiments were carried out with
their respective controls in triplicate. Eserine (0.5 mM well-1) was used as positive control. The
percent inhibition was calculated by the help of following equation.
Inhibition (%) = Control – Test x 100
Control
IC50 values were calculated using EZ–Fit Enzyme kinetics software (Perrella Scientific Inc.
Amherst, USA).
4
.6. Statistical Analysis
All the measurements were done in triplicate and statistical analysis was performed by Microsoft
Excel 2003. Results are presented as mean ± SEM.
4.7. General procedure for the synthesis of Hydrazide based Schiff bases (1-28)
The synthesis of hydrazide based Schiff bases were carried out in two steps. First methyl
salicylate (1mmol) was mixed with hydrazine hydrate (1mmol) in methanol (10 ml) as solvent
with 2-3 drops of acetic acid under continuous stirring for 4 hours to obtain hydrazide. The
reaction completion was monitored by TLC through depletion of starting material spot and
formation of hydrazide fluorescent spot established the reaction completion. After completion
distilled water was added into the reaction mixture which gives white precipitate. The product
obtained was then further purified by washing with n-hexane.
In step-2, the hydrazide was mixed with different aldehydes (1:1) in EtOH with 1-2 drops of HCl
and stirred for 2-3 hours to obtain various Schiff bases of hydrazones (1-28). The reaction
1
0

completion was checked by TLC. The Schiff bases obtained were then washed with ethanol and
n-hexane.
4
.7.1. Characterization of N'-[(Z)-(3,4-dimethoxyphenyl)methylidene]-2-hydroxybenzohydrazide
(1)
1
Yield: 0.25g (85%); m.p. 189-191 C; H-NMR (400 MHz, DMSO-d
s, 1H, -OH), 11.72 (s, 1H, -NH), 8.38 (s, 1H, N=C-H), 7.88 (d, 1H, J6,5= 7.6 Hz, H-6), 7.43 (t,
H, J4(3,5)= 7.6 Hz, H-4), 7.35 (s, 1H, H-2′), 7.23 (d, 1H, J6′,5′= 8 Hz, H-6′), 7.03 (d, 1H, J5′,6′= 8
Hz, H-5′), 3.81 (s, 3H, 3′-OCH ), 3.80 (s, 3H, 4′-OCH ); EI-MS: m/z (rel. abund. %), 301
6
) δ (ppm): 11.89
(
1
3
3
+
+
(
[M+1] , 6), 300 (M , 42.8), 180 (81), 165 (43), 163 (80), 137 (43), 121 (100), 120 (61.5), 93
56), 77 (27), 65 (77), 51 (17).
(
4
.7.2. N'-[(Z)-(2,6-dimethoxyphenyl)methylidene]-2-hydroxybenzohydrazide (2)
1
Yield: 0.12g (81%); m.p. 183-185 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 12.05
(
s, 1H, -OH), 11.72 (s, 1H, -NH), 8.61 (s, 1H, N=C-H), 7.90 (d, 1H, J6,5= 7.5 Hz, H-6), 7.42 (t,
1
H, J4(3,5)= 7.2 Hz, H-4), 7.34 (t, 1H, J4′(3′,5′)= 8.4 Hz, H-4′), 6.93 (m, 2H, H-3/ 5), 6.72 (d, 2H,
+
J
3′,4′/ 5′,4′= 8.4 Hz, H-3′/ 5′), 3.80 (s, 6H, -OCH
3
); EI-MS: m/z (rel. abund. %), 301 ([M+1] , 16.1),
+
3
00 (M , 26), 163 (44.8), 149 (100), 137 (64.3), 121 (89), 120 (65.8), 91 (37.1), 77 (13.4), 65
(26), 51 (5.4).
4.7.3. N'-{(Z)-[4-(benzyloxy)phenyl]methylidene}-2-hydroxybenzohydrazide (3)
1
Yield: 0.24g (89%); m.p. 250-252 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.90
(
s, 1H, -OH), 11.79 (s, 1H, -NH), 8.39 (s, 1H, N=C-H), 7.87 (d, 1H, J6,5= 7.5 Hz, H-6), 7.68 (d,
2
6
H, J2′,3′/ 6′,5′= 8.7 Hz, H-2′/ 6′), 7.33 (t, 1H, J4(3,5)= 6.9 Hz, H-4), 7.42 (m, 5H, H-2″/ 3″/ 4″/ 5″/
2
″), 7.10 (d, 2H, J3′,2′/ 5′/6′= 8.7 Hz, H-3′/ 5′), 6.93 (m, 2H, H-3/ 5), 5.16 (s, 2H, -OCH -Ph); EI-
+
MS: m/z (rel. abund. %), 346 (M , 0.9), 344 (10.7), 224 (16.8), 121 (9.1), 120 (9.1), 92 (14.9),
91 (100), 65 (12), 44 (37.3).
4
.7.4. 2-hydroxy-N'-[(Z)-(4-nitrophenyl)methylidene]benzohydrazide (4)
1
Yield: 0.19g (85%); m.p. 286-288 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 12.07
(
s, 1H, -OH), 11.63 (s, 1H, -NH), 8.55 (s, 1H, N=C-H), 8.31 (d, 2H, J3′,2′/ 5′,6′= 8.1 Hz, H-3′/ 5′),
.00 (d, 2H, J2′,3′/ 6′,5′= 8.4 Hz, H-2′/ 6′), 7.86 (d, 1H, J6,5= 7.2 Hz, H-6), 7.44 (t, 1H, J4(3,5)= 7.5
8
1
1

+
Hz, H-4), 6.96 (m, 2H, H-3/ 5); EI-MS: m/z (rel. abund. %), 285 (M , 3.2), 283 (5.9), 137 (13.4),
121 (100), 120 (23), 93 (13), 77 (1.8), 65 (9.7), 44 (3.6).
4
.7.5. 2-hydroxy-N'-[(Z)-(2-hydroxy-4-methoxyphenyl)methylidene]benzohydrazide (5)
1
Yield: 0.18g (80%); m.p. 259-261 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.80
(
s, 1H, -OH), 11.93 (s, 1H, -OH), 11.50 (s, 1H, -NH), 8.58 (s, 1H, N=C-H), 7.86 (d, 1H, J6,5= 7.2
Hz, H-6), 7.43 (m, 2H, H-4/ 6′), 6.95 (m, 2H, H-3/ 5), 6.52 (m, 2H, H-3′/ 5′), 3.77 (s, 3H, -
+
OCH
3
); EI-MS: m/z (rel. abund. %), 286 (M , 20), 284 (28.5), 166 (40.6), 151 (55.8), 149 (89),
121 (70.5), 120 (88.7), 92 (44.8), 91 (58), 77 (9), 65 (32), 44 (100).
4
.7.6. N'-[(Z)-(2,4-dichlorophenyl)methylidene]-2-hydroxybenzohydrazide (6)
1
Yield: 0.25g (82%); m.p. 239-241 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 12.11
(s, 1H, -OH), 11.70 (s, 1H, -NH), 8.80 (s, 1H, N=C-H), 8.02 (d, 1H, J6′,5′= 8.4 Hz, H-6′), 7.85 (d,
1
7
1
H, J6,5= 7.8 Hz, H-6), 7.73 (s, 1H, H-3′), 7.53 (d, 1H, J5′,6′= 8.6 Hz, H-5′), 7.44 (t, 1H, J4(3,5)=
+
.5 Hz, H-4), 6.95 (m, 2H, H-3/ 5); EI-MS: m/z (rel. abund. %), 310 ([M+1] , 2.22), 137 (48.6),
21 (100), 120 (58.3), 93 (40), 92 (18), 83 (34), 65 (52).
4
.7.7. N'-[(Z)-(4-butoxyphenyl)methylidene]-2-hydroxybenzohydrazide (7)
1
Yield: 0.20g (84%); m.p. 208-210 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.93
(
s, 1H, -OH), 11.74 (s, 1H, -NH), 8.38 (s, 1H, N=C-H), 7.87 (d, 1H, J6,5= 7.8 Hz, H-6), 7.66 (d,
2
3
H, J2′,3′/ 6′,5′= 8.7 Hz, H-2′/ 6′), 7.42 (t, 1H, J4(3,5)= 7.2 Hz, H-4), 7.01 (d, 2H, J3′,2′/ 5′,6′= 8.7 Hz, H-
′/ 5′), 6.94 (m, 2H, H-3/ 5), 4.01 (t, 2H, J= 6.3 Hz, -OCH
-), 0.93 (t, 3H, J= 7.35 Hz, -CH
13 ([M+1] , 3.7), 312 (M , 24), 137 (78), 136 (79), 121 (100), 120 (87), 93 (54), 77 (16), 65
2
), 1.70 (quint, 2H, J= 6.75 Hz, -CH
2
-
,
), 1.43 (sext, 2H, J= 7.3 Hz, -CH
2
3
); EI-MS: m/z (rel. abund. %),
+
+
3
(68).
4
.7.8. N'-[(Z)-(4-ethoxyphenyl)methylidene]-2-hydroxybenzohydrazide (8)
1
Yield: 0.18g (84%); m.p. 221-223 C; H-NMR (400 MHz, DMSO-d
6
) δ (ppm): 11.92
(
s, 1H, -OH), 11.74 (s, 1H, -NH), 8.39 (s, 1H, N=C-H), 7.88 (d, 1H, J6,5= 7.6 Hz, H-6), 7.67 (d,
2
3
H, J2′,3′/ 6′,5′= 8.4 Hz, H-2′/ 6′), 7.42 (t, 1H, J4(3,5)= 7.4 Hz, H-4), 7.00 (d, 2H, J3′,2′/ 5′,6′= 8.4 Hz, H-
2 3
′/ 5′), 6.94 (m, 2H, H-3/ 5), 4.08 (q, 2H, J= 7.2 Hz, -OCH -), 1.34 (t, 3H, J= 6.8 Hz, -CH ); EI-
+
+
MS: m/z (rel. abund. %), 285 ([M+1] , 6.1), 284 (M , 24.3), 164 (97), 137 (62), 136 (46), 121
100), 120 (60.4), 93 (18.7), 77 (6.8), 65 (17.3), 44 (4.1).
(
1
2

4
.7.9. N'-[(Z)-(2,4-dimethoxyphenyl)methylidene]-2-hydroxybenzohydrazide (9)
1
Yield: 0.20g (83%); m.p. 112-114 C; H-NMR (400 MHz, DMSO-d
6
) δ (ppm): 11.77
(
s, 2H, -OH, -NH), 8.71 (s, 1H, N=C-H), 7.90 (d, 1H, J6,5= 7.6 Hz, H-6), 7.80 (d, 1H, J6′,5′= 9.2
Hz, H-6′), 7.42 (t, 1H, J4(3,5)= 7.6 Hz, H-4), 6.93 (m, 2H, H-3/ 5), 6.64 (m, 2H, H-3′/ 5′), 3.86 (s,
+
3
H, 4′-OCH
3
), 3.82 (s, 3H, 2′-OCH
3
); EI-MS: m/z (rel. abund. %), 302 ([M+2] , 1.4), 301
+
+
(
[M+1] , 5.5), 300 (M , 20.1), 180 (17.6), 163 (68.5), 149 (84.7), 137 (44.3), 121 (100), 120
45), 93 (18), 77 (9.6), 65 (25), 51 (6).
(
4
.7.10. (Z)-N′-(anthracen-10-ylmethylene)-2-hydroxybenzohydrazide (10)
1
Yield: 0.23g (85%); m.p. 289-291 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 12.13
(s, 1H, -OH), 11.90 (s, 1H, -NH), 9.67 (s, 1H, N=C-H), 8.76 (m, 3H, H-1′/ 5′/ 9′), 8.17 (d, 2H,
J
4′,3′/ 6′,7′= 8.1 Hz, H-4′/ 6′), 7.98 (d, 1H, J6,5= 7.5 Hz, H-6), 7.63 (m, 4H, H-2′/ 3′/ 7′/ 8′), 7.49 (t,
+
1
H, J4(3,5)= 7.2 Hz, H-4), 7.02 (m, 2H, H-3/ 5); EI-MS: m/z (rel. abund. %), 341 ([M+1] , 2), 340
+
(
M , 11.2), 219 (34), 203 (100), 176 (22), 121 (60), 120 (18), 93 (29), 65 (43).
4.7.11. N'-[(Z)-2-furylmethylidene]-2-hydroxybenzohydrazide (11)
1
Yield: 0.11g (73%); m.p. 233-235 C; H-NMR (400 MHz, DMSO-d
6
) δ (ppm): 11.78
(s, 2H, -OH, -NH), 8.34 (s, 1H, N=C-H), 7.84 (m, 2H, H-6/ 5′), 7.42 (t, 1H, J4(3,5)= 7.6 Hz, H-4),
+
6
.94 (m,3H, H-3/ 5/ 3′), 6.64 (s, 1H, H-4′); EI-MS: m/z (rel. abund. %), 231 ([M+1] , 15.8), 230
+
(
M , 79), 137 (58), 121 (100), 120 (65), 110 (97.3), 93 (57), 65 (70), 51 (14).
4
.7.12. 2-hydroxy-N'-[(Z)-(4-isopropylphenyl)methylidene]benzohydrazide (12)
1
Yield: 0.17g (78%); m.p. 249-251 C; H-NMR (400 MHz, DMSO-d
6
) δ (ppm): 11.85
(
s, 1H, -OH), 11.79 (s, 1H, -NH), 8.42 (s, 1H, N=C-H), 7.88 (d, 1H, J6,5= 7.6 Hz, H-6), 7.66 (d,
2
5
2
H, J2′,3′/ 6′,5′= 8 Hz, H-2′/ 6′), 7.43 (t, 1H, J4(3,5)= 7.6 Hz, H-4), 7.34 (d, 2H, J3′,2′/ 5′,6′= 8 Hz, H-3′/
+
′), 2.93 (m, 1H, -CH-), 1.21 (d, 6H, J= 6.8 Hz, -CH
3
); EI-MS: m/z (rel. abund. %), 282 (M ,
0.7), 162 (83), 147 (76.7), 137 (75), 121 (100), 120 (73.2), 93 (18), 65 (19.6).
4
.7.13. N'-{(Z)-[4-(tert-butyl)phenyl]methylidene}-2-hydroxybenzohydrazide (13)
1
Yield: 0.18g (78%); m.p. 245-246 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.85
(
s, 1H, -OH), 11.79 (s, 1H, -NH), 8.42 (s, 1H, N=C-H), 7.87 (d, 1H, J6,5= 7.5 Hz, H-6), 7.67 (d,
2
H, J2′,3′/ 6′,5′= 8.1 Hz, H-2′/ 6′), 7.49 (d, 2H, J3′,2′/ 5′,6′= 8.1 Hz, H-3′/ 5′), 7.43 (t, 1H, J4(3,5)= 7.5
1
3

+
3
Hz, H-4), 6.94 (m, 2H, H-3/ 5), 1.29 (s, 9H, -CH ); EI-MS: m/z (rel. abund. %), 296 (M , 22.4),
176 (38), 162 (12.4), 161 (79.7), 137 (72.5), 121 (100), 120 (64), 93 (13.4), 91 (11), 65 (16).
4
.7.14. 2-hydroxy-N'-{(Z)-[3-(trifluoromethyl)phenyl]methylidene}benzohydrazide (14)
1
Yield: 0.15g (83%); m.p. 227-229 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.99
(s, 1H, -OH), 11.70 (s, 1H, -NH), 8.53 (s, 1H, N=C-H), 8.07 (s, 1H, H-2′), 8.04 (d, 1H, J6′,5′= 8.4
Hz, H-6′), 7.87 (d, 1H, J6,5= 7.5 Hz, H-6), 7.80 (d, 1H, J4′,5′= 7.8 Hz, H-4′), 7.71 (t, 1H, J5′(4′,6′)
=
7
.5 Hz, H-5′), 7.44 (t, 1H, J4(3,5)= 7.2 Hz, H-4), 6.96 (m, 2H, H-3/ 5); EI-MS: m/z (rel. abund.
+
+
%
), 309 ([M+1] , 2.8), 308 (M , 18), 137 (30.79), 122 (31.4), 121 (100), 120 (37.4), 93 (40.3),
65 (58), 51 (11.8).
4.7.15. N'-[(Z)-(2,3-dimethoxyphenyl)methylidene]-2-hydroxybenzohydrazide (15)
1
Yield: 0.19g (80%); m.p. 182-184 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.91
(
s, 2H, -OH, -NH), 8.73 (s, 1H, N=C-H), 7.89 (d, 1H, J6,5= 7.5 Hz, H-6), 7.46 (m, 2H, H-4/ 6′),
.13 (m, 2H, H-4′/ 5′), 6.94 (m, 2H, H-3/ 5), 3.83 (s, 3H, 2′-OCH ), 3.80 (s, 3H, 3′-OCH ); EI-
MS: m/z (rel. abund. %), 301 ([M+1] , 4.8), 300 (M , 36), 163 (64.7), 149 (74.3), 137 (54.2),
7
3
3
+
+
121 (100), 120 (65), 93 (52), 65 (73), 51 (11).
4
.7.16. N'-{(Z)-[3-(benzyloxy)-4-methoxyphenyl]methylidene}-2-hydroxybenzohydrazide (16)
1
Yield: 0.24g (81%); m.p. 203-205 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.90
(
s, 1H, -OH), 11.75 (s, 1H, -NH), 8.36 (s, 1H, N=C-H), 7.87 (d, 1H, J6,5= 7.5 Hz, H-6), 7.49 (m,
H, H-6′), 7.44 (m, 3H, H-4/ 2″/ 6″), 7.40 (s, 1H, H-2′), 7.32 (m, 3H, H-3″/ 4″/ 5″), 7.07 (d, 1H,
5′,6′= 8.4 Hz, H-5′), 6.94 (m, 2H, H-3/ 5), 5.13 (s, 2H, -OCH -Ph), 3.82 (s, 3H, -OCH ); EI-MS:
m/z (rel. abund. %), 377 ([M+1] , 3.6), 376 (M , 19.5), 165 (11), 137 (13.5), 121 (60), 91 (100),
1
J
2
3
+
+
65 (42).
4
.7.17. N'-[(Z)-(3-bromo-4-methoxyphenyl)methylidene]-2-hydroxybenzohydrazide (17)
1
Yield: 0.23g (85%);m.p. 216-218 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.82
(
(
s, 2H, -OH, -NH), 8.36 (s, 1H, N=C-H), 7.96 (s, 1H, H-2′), 7.87 (d, 1H, J6,5= 7.5 Hz, H-6), 7.72
d, 1H, J6′,5′= 7.2 Hz, H-6′), 7.43 (t, 1H, J4(3,5)= 7.2 Hz, H-4), 7.21 (d, 1H, J5′,6′= 8.4 Hz, H-5′),
+
6
.94 (m, 2H, H-3/ 5), 3.90 (s, 3H, -OCH
3
); EI-MS: m/z (rel. abund. %), 351 ([M+2] , 3.8), 350
+
+
(
[M+1] , 9.2), 349 (M , 3.4), 230 (76.3), 228 (65), 137 (64), 121 (100), 120 (35), 93 (19), 65
40).
(
1
4

4
.7.18.2-hydroxy-N'-{(Z)-[4-(trifluoromethyl)phenyl]methylidene}benzohydrazide (18)
1
Yield: 0.18g (76%); m.p. 272-274 C; H-NMR (400 MHz, DMSO-d
6
) δ (ppm): 11.97
(
s, 1H, -OH), 11.68 (s, 1H, -NH), 8.52 (s, 1H, N=C-H), 7.96 (d, 2H, J3′,2′/ 5′,6′= 8 Hz, H-3′/ 5′),
.87 (d, 1H, J6,5= 7.6 Hz, H-6), 7.82 (d, 2H, J2′,3′/ 6′,5′= 8.4 Hz, H-2′/ 6′), 7.44 (t, 1H, J4(3,5)= 7.6
7
+
+
Hz, H-4), 6.96 (m, 2H, H-3/ 5); EI-MS: m/z (rel. abund. %), 309 ([M+1] , 5.1), 308 (M , 20),
137 (29.4), 122 (28), 121 (100), 120 (37), 93 (37.7), 65 (51), 51 (12.7).
4
.7.19. N'-[(Z)-(3-bromophenyl)methylidene]-2-hydroxybenzohydrazide (19)
1
Yield: 0.19g (94%); m.p. 241-243 C; H-NMR (400 MHz, DMSO-d
6
) δ (ppm): 11.92
(
s, 1H, -OH), 11.72 (s, 1H, -NH), 8.42 (s, 1H, N=C-H), 7.93 (s, 1H, H-2′), 7.87 (d, 1H, J6,5= 7.6
Hz, H-6), 7.73 (d, 1H, J6′,5′= 7.6 Hz, H-6′), 7.63 (d, 1H, J4′,5′= 8 Hz, H-4′), 7.43 (m, 2H, H-4/ 5′),
+
+
6
.95 (m, 2H, H-3/ 5); EI-MS: m/z (rel. abund. %), 321 ([M+2] , 8.7), 320 ([M+1] , 36.6), 319
+
(
M , 10), 318 (45), 200 (26.3), 137 (80), 121 (100), 120 (84), 93 (60), 65 (54).
4.7.20. N'-[(Z)-(2-ethoxyphenyl)methylidene]-2-hydroxybenzohydrazide (20)
1
Yield: 0.27g (94%);m.p. 194-196 C; H-NMR (400 MHz, DMSO-d
6
) δ (ppm): 11.98
(
s, 1H, -OH), 11.91 (s, 1H, -NH), 8.80 (s, 1H, N=C-H), 7.89 (d, 2H, J6,5/ 6′,5′= 8.8 Hz, H-6/ 6′),
7
2
2
6
.42 (m, 2H, H-4/ 4′), 7.09 (d, 1H, J3′,4′= 8 Hz, H-3′), 7.01 (t, 1H, J5′(4′,6′)= 7.4 Hz, H-5′), 6.93 (m,
+
H, H-3/ 5), 4.13 (q, 2H, -OCH
2
-), 1.39 (t, 3H, -CH
3
); EI-MS: m/z (rel. abund. %), 284 (M ,
3.5), 148 (10), 137 (71), 133 (84.6), 121 (100), 120 (80), 105 (25), 93 (20.4), 91 (11.3), 77 (9),
5 (26.3).
4
.7.21. N'-[(Z)-(2-bromo-5-fluorophenyl)methylidene]-2-hydroxybenzohydrazide (21)
1
Yield: 0.26g (78%); m.p. 233-235 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 12.20
(s, 2H, -OH, -NH), 8.76 (s, 1H, N=C-H), 7.84 (d, 1H, J6,5= 7.5 Hz, H-6), 7.73 (m, 2H, H-3′/ 6′),
7
.44 (t, 1H, J4(3,5)= 7.5 Hz, H-4), 7.29 (t, 1H, J4′,3′= 8 Hz, H-4′), 6.96 (m, 2H, H-3/ 5); EI-MS: m/z
+
+
+
(
rel. abund. %), 339 ([M+2] , 2.1), 338 ([M+1] , 6), 337 (M , 2.3), 258 (13.8), 137 (89.5), 121
100), 120 (91.4), 93 (41), 65 (44).
(
4.7.22.2-hydroxy-N'-{(Z)-[4-(methylsulfanyl)phenyl]methylidene}benzohydrazide (22)
1
Yield: 0.21g (73%); m.p. 259-261 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.81
(
s, 1H, -OH), 11.78 (s, 1H, -NH), 8.40 (s, 1H, N=C-H), 7.87 (d, 1H, J6,5= 7.2 Hz, H-6), 7.67 (d,
2
H, J2′,3′/ 6′,5′= 8.1 Hz, H-2′/ 6′), 7.43 (t, 1H, J4(3,5)= 6.9 Hz, H-4), 7.33 (d, 2H, J3′,2′/ 5′,6′= 8.4 Hz, H-
1
5

+
3
2
7
3
′/ 5′), 6.95 (m, 2H, H-3/ 5), 2.51 (s, 3H, -SCH ); EI-MS: m/z (rel. abund. %), 288 ([M+2] ,
+
+
.6), 287 ([M+1] , 9), 286 (M , 42), 166 (75), 149 (56), 137 (51), 121 (100), 120 (56), 93 (56),
7 (38), 65 (80), 51 (24).
4
.7.23. 2-hydroxy-N'-[(Z)-2-thienylmethylidene]benzohydrazide (23)
1
Yield: 0.17g (83%); m.p. 252-254 C; H-NMR (400 MHz, DMSO-d
6
) δ (ppm): 11.77
(
s, 2H, -OH, -NH), 8.66 (s, 1H, N=C-H), 7.84 (d, 1H, J6,5= 8 Hz, H-6), 7.69 (d, 1H, J5′,4′= 4.8 Hz,
H-5′), 7.49 (d, 1H, J3′,4′= 3.2 Hz, H-3′), 7.43 (t, 1H, J4(3,5)= 7.2 Hz, H-4), 7.15 (t, 1H, J4′(3′,5′)= 4
+
+
Hz, H-4′), 6.94 (m, 2H, H-3/ 5); EI-MS: m/z (rel. abund. %), 248 ([M+2] , 3.1), 247 ([M+1] ,
+
1
0.3), 246 (M , 28.6), 137 (67), 126 (94), 121 (100), 120 (57), 93 (34), 65 (40).
4.7.24. N'-[(Z)-(3,5-dichloro-2-hydroxyphenyl)methylidene]-2-hydroxybenzohydrazide (24)
1
Yield: 0.24g (75%);m.p. 265-267 C; H-NMR (400 MHz, DMSO-d
6
) δ (ppm): 12.40
(
s, 1H, -OH), 12.30 (s, 1H, -OH), 11.52 (s, 1H, -NH), 8.61 (s, 1H, N=C-H), 7.86 (d, 1H, J6,5= 7.2
Hz, H-6), 7.64 (s, 1H, H-6′), 7.63 (s, 1H, H-4′), 7.46 (t, 1H, J4(3,5) = 8 Hz, H-4), 6.98 (m, 2H, H-3/
+
5
); EI-MS: m/z (rel. abund. %), 326 ([M+1] , 30), 324 (39), 204 (22), 137 (57), 121 (100), 120
(62), 93 (33), 65 (32).
4
.7.25. N'-[(Z)-(3-bromo-4-fluorophenyl)methylidene]-2-hydroxybenzohydrazide (25)
1
Yield: 0.27g (80%); m.p. 254-256 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.94
(
s, 1H, -OH), 11.74 (s, 1H, -NH), 8.41 (s, 1H, N=C-H), 8.06 (d. 1H, J2′,6′= 5.7 Hz, H-2′), 7.86 (d,
1
H, J6,5= 7.5 Hz, H-6), 7.80 (t, 1H, J6′(2′,5′)= 5.8 Hz, H-6′), 7.49 (d, 1H, J5′,6′= 8.7 Hz, H-5′), 7.43
+
(
t, 1H, J4(3,5)= 6.9 Hz, H-4), 6.95 (m, 2H, H-3/ 5); EI-MS: m/z (rel. abund. %), 339 ([M+2] ,
+
+
2
.1), 338 ([M+1] , 6), 337 (M , 2.3), 216 (8), 137 (97), 122 (42), 121 (100), 120 (59.4), 107
(33), 93 (45), 65 (73), 57 (10).
4
.7.26. N'-[(Z)-(4-bromo-2-fluorophenyl)methylidene]-2-hydroxybenzohydrazide (26)
1
Yield: 0.28g (85%); m.p. 259-261 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 12.00
(
s, 1H, -OH), 11.69 (s, 1H, -NH), 8.62 (s, 1H, N=C-H), 7.87 (m, 2H, H-6/ 3′), 7.69 (d, 1H, J6′,5′
=
10.2 Hz, H-6′), 7.53 (d, 1H, J5′,6′= 8.4 Hz, H-5′), 7.44 (t, 1H, J4(3,5)= 7.2 Hz, H-4), 6.95 (m, 2H,
+
+
+
H-3/ 5); EI-MS: m/z (rel. abund. %), 339 ([M+2] , 1.3), 338 ([M+1] , 9.3), 337 (M , 1.2), 218
10.4), 137 (49.7), 121 (100), 120 (57), 107 (31), 93 (50), 65 (68), 57 (25).
(
1
6

4
.7.27. 2-hydroxy-N'-[(Z)-(3-hydroxyphenyl)methylidene]benzohydrazide (27)
1
Yield: 0.16g (78%); m.p. 253-255 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.84
(s, 1H, -OH), 11.78 (s, 1H, -NH), 9.64 (s, 1H, -OH), 8.36 (s, 1H, N=C-H), 7.87 (d, 1H, J6,5= 7.8
Hz, H-6), 7.43 (t, 1H, J4(3,5)= 7.2 Hz, H-4), 7.25 (t, 1H, J5′(4′,6′)= 7.5 Hz, H-5′), 7.20 (s, 1H, H-2′),
+
6
5
.95 (m, 2H, H-3/ 5), 6.83 (d, 1H, J4′,5′= 7.2 Hz, H-4′); EI-MS: m/z (rel. abund. %), 257 ([M+1] ,
+
.3), 256 (M , 19.6), 137 (57), 136 (31), 121 (100), 120 (62.7), 93 (26), 77 (12.4), 65 (47), 51
(10).
4
.7.28.2-hydroxy-N'-[(Z)-(4-hydroxyphenyl)methylidene]benzohydrazide (28)
1
Yield: 0.19g (77%); m.p. 239-241 C; H-NMR (300 MHz, DMSO-d
6
) δ (ppm): 11.98
(
s, 1H, -OH), 11.67 (s, 1H, -NH), 9.96 (s, 1H, -OH), 8.34 (s, 1H, N=C-H), 7.87 (d, 1H, J6,5= 7.5
Hz, H-6), 7.57 (d, 2H, J2′,3′/ 6′,5′= 8.4 Hz, H-2′/ 6′), 7.42 (t, 1H, J4(3,5)= 7.2 Hz, H-4), 6.95 (m, 2H,
+
H-3/ 5), 6.83 (d, 2H, J3′,2′/ 5′,6′= 8.4 Hz, H-3′/ 5′); EI-MS: m/z (rel. abund. %), 257 ([M+1] , 5),
+
2
56 (M , 19.8), 137 (63), 136 (87), 121 (100), 120 (63.8), 93 (26), 77 (13), 65 (50), 51 (11).
1
7

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Kaapro,
J.
Ojanen;
Protein
docking.
(2002).
Available
at
4
(
2
0

Captions:
Scheme-1: Synthesis of hydrazide based Schiff bases.
Table-1: Different Substituent of Schiff base of Salicyloyl hydrazide analogs 1-28
Table-2: Acetylcholinesterase and Butyrylcholinesterase activity of compounds 1-28
Figure 1: (A) 3D binding mode of compound 10 and (B) 2D image of compound 10.
Figure 2: (A) 3D conformation of compound 3 and (B) 2D interaction of compound 3 with
residues of ACHE.
Figure 3: (A) 3D interaction image of compound 24 and (B) 2D interaction of compound 24
with active amino acid residues of ACHE.
Figure 4: (A) 3D binding mode of compound 1 and (B) 2D interaction image of compound 1.
Figure 5: (A) 3D interaction of compound 13 with BCHE and (B) 2D image of compound 13.
Figure 6: (A) 3D interaction of compound 24 with BCHE and (B) 2D image of compound 24.
Figure 7: (A) 3D interaction image of compound 3 and (B) 2D interaction of compound 3 with
active amino acid residues of BCHE.
Figure 8: (A) 3D binding mode of compound 1 and (B) 2D binding mode of compound 1.
Scheme:
2
1

Figure 1: (A) 3D binding mode of compound 10 and (B) 2D image of compound 10.
Figure 2: (A) 3D conformation of compound 3 and (B) 2D interaction of compound 3 with
residues of ACHE.
2
2

Figure 3: (A) 3D interaction image of compound 24 and (B) 2D interaction of compound 24
with active amino acid residues of ACHE.
Figure 4: (A) 3D binding mode of compound 1 and (B) 2D interaction image of compound 1.
2
3

Figure 5: (A) 3D interaction of compound 13 with BCHE and (B) 2D image of compound 13.
Figure 6: (A) 3D interaction of compound 24 with BCHE and (B) 2D image of compound 24.
2
4

Figure 7: (A) 3D interaction image of compound 3 and (B) 2D interaction of compound 3 with
active amino acid residues of BCHE.
Figure 8: (A) 3D binding mode of compound 1 and (B) 2D binding mode of compound 1.
2
5

Table-1: Different Substituent of Schiff base of Salicyloyl hydrazide analogs 1-28
S.
S. No
R
R
S. No
R
No
11
1
21
2
12
22
3
4
5
13
14
15
23
24
25
6
7
16
17
26
27
8
9
18
19
28
2
6

1
0
20
Table-2: Acetylcholinesterase and Butyrylcholinesterase activity of compounds 1-28
Serial
Inhibition (%) at 0.5
mM for AChE
20.57±0.34
Inhibition (%) at 0.5
mM for BChE
34.11±0.41
IC50 (µM)
IC50 (µM)
#
1
2
NA
461.3±0.17
NA
79.51±0.13
52.45±0.22
73.67±0.26
3
92.23±0.11
8.12±0.01
85.46±0.33
37.82±0.14
4
5
6
7
8
9
42.71±0.44
38.71±0.35
85.42±0.26
86.21±0.24
58.34±0.35
27.71±0.37
94.22±0.15
17.81±0.51
84.17±0.23
68.22±0.25
78.45±0.31
31.89±0.28
83.29±0.17
89.74±0.13
79.34±0.22
62.15±0.28
53.67±0.21
55.34±0.37
52.55±0.35
54.37±0.24
93.45±0.17
54.27±0.33
57.78±0.31
56.33±0.29
46.57±0.24
82.82±1.09
NA
NA
23.44±0.31
13.42±0.29
83.15±0.22
82.78±0.23
61.45±0.27
53.56±0.21
28.89±0.33
52.37±0.21
75.45±0.15
93.45±0.07
62.44±0.17
76.23±0.17
72.36±0.21
81.09±0.11
55.27±0.18
62.67±0.16
63.69±0.18
52.16±0.22
10.56±0.31
40.78±0.39
94.34±0.21
33.28±0.28
54.22±0.32
34.22±0.44
52.13±0.27
91.29±1.17
NA
NA
18.12±0.02
15.37±0.03
217.7±0.28
NA
4.12±0.01
NA
39.81±0.12
92.43±0.19
56.13±0.14
NA
18.31±0.02
19.81±0.05
48.51±0.16
155.92±0.22
415.27±0.14
311.53±0.27
435.68±0.26
429.84±0.19
8.41±0.06
311.42±0.21
369.15±0.25
354.92±0.14
NA
43.15±0.15
41.81±0.16
116.3±0.14
293.2±0.13
NA
417.92±0.11
71.81±0.13
6.51±0.01
112.23±0.15
61.64±0.18
86.21±0.16
38.41±0.13
213.25±0.15
121.32±0.12
116.15±0.19
267.42±0.14
NA
NA
9.22±0.07
NA
451.6±0.21
NA
423.52±0.12
0.04±0.0001
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Eserine
0.85±0.0001
2
7

Synthesis and in vitro Acetylcholinesterase and Butyrylcholinesterase Inhibitory Potential
of Hydrazide based Schiff Bases
a∗
a
b,c
d
a
Fazal Rahim, Hayat Ullah, Muhammad Taha, Abdul Wadood, Muhammad Tariq Javed, Wajid
a
a
e
f
g
Rehman , Mohsan Nawaz, Muhammad Ashraf , Muhammad Ali , Muhammad Sajid , Farman Ali,
a
h
Muhammad Naseem Khan, Khalid Mohammed Khan
a
b
Depatment of Chemistry, Hazara University, Mansehra-21300, Pakistan. Atta-ur-Rahman Institute for Natural Product
Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia,
c
d
Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia. Department of Biohemistry, Abdul Wali Khan
e
University Mardan, Mardan-23200, Pakistan. Department of Biochemistry and Biotechnology, The Islamia University of
f
Bahawalpur, Bahawalpur 63100. Pakistan. Center for Advanced Drug Research, COMSATS Institute of Information
g
Technology, University Road, Abbottabad 22060, KPK, Pakistan. Department of Biochemistry, Hazara University, Mansehra-
h
2
1300, Pakistan. H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi-75270, Pakistan.
*
Corresponding Author: fazalstar@gmail.com, Tel.: 0092-335-9528343;
2
8

Highlights:
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Synthesis of hydrazide based Schiff bases derivatives
In vitro acetylcholinesterase and butyrylcholinesterase inhibitory activity
Identification of a new class of acetylcholinesterase and butyrylcholinesterase inhibitors
Structure-activity Relationship established
Molecular docking
2
9