Novel phenolic Mannich base derivatives: synthesis, bioactivity, molecular docking, and ADME-Tox Studies

Article abstract of DOI:10.1007/s13738-021-02331-8

In this study, it was aimed to synthesize novel molecules containing potential biological active phenolic Mannich base moiety and evaluate the inhibition properties against α-glycosidase (α-Gly) and acetylcholinesterase (AChE). For this purpose, phenolic aldehydes (1–3) were synthesized from 4-hydroxy-3-methoxy benzaldehyde (vanillin) according to the Mannich Reaction. Five different carboxylic acid hydrazides (4a-e) were synthesized from esters obtained from carboxylic acids. Fifteen Schiff base derivatives (5a-e, 6a-e, and 7a-e) were synthesized from the condensation reaction of compounds 1–3 with 4a-e. In this work, a series of novel Schiff bases from Phenolic Mannich bases (5a-e, 6a-e, and 7a-e) were tested toward α-Gly and AChE enzymes. Compounds 5a-e, 6a-e, and 7a-e showed Kis in ranging of 341.36 ± 31.84–904.76 ± 93.56?nM on AChE and 176.27 ± 22.87—621.77 ± 69.98?nM on α-glycosidase. Finally, novel compounds were found using molecular docking method to calculate the biological activity of these bases against many enzymes. The enzymes used in these calculations are acetylcholinesterase and α-glycosidase, respectively. Molecule 6b is more effective and active than other molecules with a docking score parameter value of ? 8.77 against AChE enzyme and 6d is more effective and active than other molecules with a docking score parameter value of ? 4.94 against α-Gly enzyme. After calculating the biological activities of novel compounds, ADME/T analysis parameters were examined to calculate the future drug use properties.

Full text of DOI:10.1007/s13738-021-02331-8

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-021-02331-8
ORIGINAL PAPER
Novel phenolic Mannich base derivatives: synthesis, bioactivity,
molecular docking, and ADME‑Tox Studies
Feyzi Sinan Tokalı1 · Parham Taslimi2 · İbrahim Hakkı Demircioğlu3 · Kıvılcım Şendil4 · Burak Tuzun5 · İlhami Gülçin3
Received: 10 May 2021 / Accepted: 25 June 2021
© Iranian Chemical Society 2021
Abstract
In this study, it was aimed to synthesize novel molecules containing potential biological active phenolic Mannich base moi-
ety and evaluate the inhibition properties against α-glycosidase (α-Gly) and acetylcholinesterase (AChE). For this purpose,
phenolic aldehydes (1–3) were synthesized from 4-hydroxy-3-methoxy benzaldehyde (vanillin) according to the Mannich
Reaction. Five diferent carboxylic acid hydrazides (4a-e) were synthesized from esters obtained from carboxylic acids. Fif-
teen Schif base derivatives (5a-e, 6a-e, and 7a-e) were synthesized from the condensation reaction of compounds 1–3 with
4a-e. In this work, a series of novel Schif bases from Phenolic Mannich bases (5a-e, 6a-e, and 7a-e) were tested toward α-Gly
and AChE enzymes. Compounds 5a-e, 6a-e, and 7a-e showed Kis in ranging of 341.36 31.84–904.76 93.56 nM on AChE
and 176.27 22.87—621.77 69.98 nM on α-glycosidase. Finally, novel compounds were found using molecular docking
method to calculate the biological activity of these bases against many enzymes. The enzymes used in these calculations
are acetylcholinesterase and α-glycosidase, respectively. Molecule 6b is more efective and active than other molecules with
a docking score parameter value of−8.77 against AChE enzyme and 6d is more efective and active than other molecules
with a docking score parameter value of−4.94 against α-Gly enzyme. After calculating the biological activities of novel
compounds, ADME/T analysis parameters were examined to calculate the future drug use properties.
Keywords Phenolic Mannich bases · Schif bases · Enzyme inhibition · Molecular docking
Introduction
various practical applications such as paint, cosmetics, the
improvement of natural macromolecular materials, analyti-
cal reagents, water treatment products, petroleum additives,
synthetic polymer products, textiles, leather, and paper [1].
However, the most important application area of Mannich
bases is pharmaceutical chemistry and this claim is sup-
ported by a signifcant number of papers published on this
subject every year. The Mannich reaction can be used to
improve the distribution of drugs in the human body. Hydro-
philic property of the drug can be increased by inserting a
polar group into the structure by Mannich reaction. In addi-
tion, if the appropriate amine reagent is used in the Mannich
reaction, the lipophilic property of the drug can be increased
[2]. In addition, aminomethylated drugs can act as prodrugs,
by releasing the active part with deaminomethylation [3] or
deamination [4] under controlled hydrolytic conditions. Phe-
nolic Mannich bases are in the group of C-Mannich bases
and represent a very important class of compounds due to
the formation of the C–C bond and their biological activi-
ties. There are many studies in the literature that phenolic
Mannich bases show important biological activities such as
Mannich reaction is a condensation reaction between active
hydrogen compounds, an aldehyde, and an amine, lead-
ing to the formation of Mannich bases. Mannich bases are
generally classifed as C-Mannich bases, N-Mannich bases,
S-Mannich bases, and P-Mannich bases. Mannich bases have
* Feyzi Sinan Tokalı
feyzitokali@gmail.com; feyzitokali@kafkas.edu.tr
1
Kars Vocational School, Department of Material
and Material Processing Technologies, Kafkas University,
36100 Kars, Turkey
2
Department of Biotechnology, Faculty of Science, Bartin
University, 74100 Bartin, Turkey
3
Department of Chemistry, Faculty of Sciences, Ataturk
University, 25000 Erzurum, Turkey
4
Department of Chemistry, Faculty of Arts and Sciences,
Kafkas University, 36100 Kars, Turkey
5
Department of Chemistry, Faculty of Science, Cumhuriyet
University, 58140 Sivas, Turkey
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
anticancer and cytotoxic activity [5–7], antibacterial activity
[8, 9], antimycobacterial activity [10, 11], antifungal activity
[12, 13], antiviral activity [14–16], anti-hepatitis-B activity
[17, 18], anticonvulsant activity [19, 20], anti-infammatory
activity [21, 22], analgesic activity [23], antioxidant [24,
25], enzyme inhibition activity [26], regulation of blood
pressure [27, 28], and platelet aggregation inhibitory activ-
ity [29].
theoretically calculates the efects of drug molecules on dif-
ferent organs and tissues. It is theoretically known whether
these drug molecules will be more efective by applying
them to the skin, taking them orally, or as a vaccine.
In this study, it was aimed to synthesize new Schif bases
from Phenolic Mannich bases and test the enzyme inhibition
activities against α-Gly and AChE and molecular docking
studies.
Type-2 diabetes mellitus (T2DM) is one of the most
popular metabolic diseases in the world and is defned by
hyperglycemia [30, 31]. α-Glycosidase enzymes are in the
nutritive system that hydrolyzes carbohydrate molecules into
glucose molecules. One mechanism that has been developed
to therapy of T2DM is inhibition of α-glycosidase enzymes
using synthetic drugs [32, 33]. Acetylcholinesterase (AChE)
is an enzyme present in cholinergic neurons, and its key role
is the rapid breakdown of the neurotransmitter acetylcholine
(ACh) released throughout neurotransmission. The level of
the active site of AChE is a 20 Å length gorge that includes
an anionic catalytic site and a peripheral anionic binding
site. Additionally, AChE inhibitor compounds modulate
ACh hydrolysis and also play an important role in diagnos-
ing the cholinergic tone [34–37].
Result and discussion
Chemistry
Phenolic aldehydes (1–3) were synthesized according to the
Mannich Reaction. Carboxylic acid hydrazides (4a-e) were
synthesized from esters obtained from suitable carboxylic
acids according to Fischer Esterifcation. In the last step phe-
nolic aldehydes (1–3) were reacted with hydrazides (4a-e)
and target compounds (5a-e, 6a-e, and 7a-e) were obtained
with good yields (79–93%). Structures of new compounds
were characterized by IR, 1H NMR, 13C NMR, and HRMS
spectroscopic methods.
When many studies are examined today, it is seen that
theoretical and experimental studies are carried out together.
It is seen that the studies done in this way are more popular.
When these studies are examined, it is seen that the results
of experimental and theoretical calculations are very close
to each other [38–43]. Therefore, the theoretical calcula-
tions made before the experimental procedures guide the
experimental procedures. With theoretical calculations, it is
possible to synthesize more efective and active molecules
with their results. In this study, the best method to com-
pare the biological activities of novel compounds against
enzymes for theoretical calculations is molecular docking.
In the calculations made by molecular docking method, the
biological activity values of the molecules found as a result
of calculations made against enzymes of compounds 5a-e,
6a-e, and 7a-e are estimated. It is possible to compare the
biological activities of other molecules with the numerical
values obtained as a result of the calculation [44, 45]. Many
parameters are obtained by molecular docking calculations
of novel molecules against enzymes. From these parameters
obtained from molecular docking calculations, a lot of infor-
mation about the biological activities of novel molecules is
obtained [46]. After these calculations, ADME/T (Absorp-
tion, Distribution, Metabolism, Excretion, and Toxicity)
analysis of novel molecules was performed. ADME/T analy-
sis aims to predict the efects and reactions of molecules that
can be drugs on the human body [47, 48]. These molecules
have many parameters with ADME/T calculations. These
efects and responses are tried to be predicted with numeri-
cal values of these parameters. Each parameter calculated
In the IR spectra of the compounds 5a-e, 6a-e, and 7a-e,
NH stretching bands are observed at 3273–3273 cm−1.
C = O and CH = N stretching bands are observed at
1670–1626 cm−1 and 1611–1583 cm−1, respectively. NH,
C = O, and CH = N stretching bands are the characteristic
bands of the compounds and compatible with structures.
1
In the H NMR spectra of the compounds 5a-e, 6a-e,
and 7a-e, peaks of NH protons are seen as a singlet at δ
12.05–10.20 ppm. Peaks of N = CH protons are observed
as singlet at δ 8.75–8.19 ppm. Aromatic proton peaks are
observed at δ 8.43–6.79 ppm as singlet, doublet, triplet,
and multiplet relative to their chemical environment. Peaks
of OCH3 and Ph-CH2-N protons are seen as a singlet at δ
3.98–3.79 and δ 3.85–3.61 ppm, respectively. Aliphatic pro-
tons of morpholine, piperazine, and piperidine moieties are
observed at δ 3.78–0.80 ppm as doublet, triplet, and multi-
plet relative to their chemical environment. In the 1H NMR
spectra of compounds containing phenoxyacetyl moiety (5d,
6d, and 7d), peaks of NH, N=CH, and Ph–O–CH2 protons
are seen as two singlets due to E/Z isomers and cis/trans
conformers of the molecules [49–51].
In the 13C NMR spectra of fnal molecules, peaks of C=O
and CH=N carbons are seen at δ 166.8–160.7 ppm and δ
149.9–147.4 ppm, respectively. Peaks of aromatic carbons
are observed at δ 168.8–108.1 ppm. Peaks of Ph–O–CH2
and OCH3 carbons are seen at δ 61.4–57.9 ppm and δ
57.5–55.5 ppm, respectively. And aliphatic carbons of mor-
pholine, piperazine, and piperidine moieties are observed at
δ 66.7–19.2 ppm.
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Journal of the Iranian Chemical Society
Finally, in the HRMS spectra of target compounds, the
found M+1 molecular ion values are compatible with the
calculated M+1 molecular ion values. Spectral data of the
compounds are fully compatible with the structure of the
molecules.
and α-glycosidase as described in previous methods. The
inhibitory activities were compared to standard compounds
(Table 1 and Fig. 1). The following results were recorded:
These cholinergic enzyme inhibition results are reported
in Table 1. Compounds 5a-e, 6a-e, and 7a-e had Kis in
ranging from 341.36 31.84 to 904.76 93.56 nM for
AChE (Table 1 and Fig. 1). In comparison, TAC had Ki
of 787.25 80.61 nM against indicated AChE enzyme. It
could be seen from the table that all novel molecules dem-
onstrated marked inhibitory effects against AChE with
Bioactivity
The enzyme inhibitory efects of all the novel Schif bases
(5a-e, 6a-e, and 7a-e) were investigated against AChE
Table 1 The enzyme inhibition
Compounds
IC50 (nM)
AChE
Ki (nM)
r2
results of compounds
5a-e, 6a-e, 7a-e against
achethylcholinesterase (AChE)
and α-glycosidase (α-Gly)
enzymes
r2
α-Gly
AChE
α-Gly
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
TAC**
ACR***
1094.27
502.88
592.67
632.51
600.42
835.02
398.46
445.90
549.25
513.86
1015.74
802.53
841.38
943.26
813.44
848.84
–
0.9781
0.9889
0.9610
0.9937
0.9614
0.9637
0.9041
0.9382
0.9901
0.9779
0.9472
0.9284
0.9620
0.9399
0.9692
0.9668
–
411.28
193.24
244.06
212.88
200.74
318.10
201.52
155.37
192.66
197.13
612.92
504.06
441.57
341.48
368.01
–
0.9558
0.9972
0.9921
0.9328
0.9660
0.9031
0.9715
0.9183
0.9912
0.9551
0.9621
0.9815
0.9228
0.9293
0.9815
–
845.15 56.01
441.88 29.07
504.81 50.01
553.94 66.17
498.06 43.06
779.21 82.80
341.36 31.84
401.96 38.87
491.55 49.77
455.15 46.93
904.76 93.56
698.15 72.08
735.85 81.04
879.33 90.02
711.47 59.72
787.25 80.61
–
476.13 60.32
238.03 26.95
278.03 33.18
226.98 37.92
254.14 50.83
354.83 26.96
214.04 53.82
176.27 22.87
200.15 48.51
209.45 26.17
621.77 69.98
557.13 39.67
478.32 67.90
359.62 17.86
395.03 47.01
–
513.41
0.9491
548.13 62.38
*(They are control compounds)
Fig. 1 Ki values of AChE and
1000
900
800
700
600
500
400
300
200
100
0
Ki values (nM)
α-glycosidase enzymes
AChE
α-Gly
TAC, 5a
ACR
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
Compounds
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Journal of the Iranian Chemical Society
Ki values ranging in sub-nanomolar; however, compound
6b showed perfect inhibition effect against AChE (Ki:
341.36 31.84 nM; Ki-TAC/Ki-6b: 2.28) (Table 1). For
AChE, IC50 values of TAC as positive control and some
novel compounds the following order: 6b (398.46 nM, r2:
0.9041)<6c (445.90 nM, r2: 0.9382)<5b (502.88 nM, r2:
0.9882)<6e (513.86 nM, r2: 0.9779)<TAC (848.84 nM, r2:
0.9668). Cholinergic neurotransmission plays a key role in
impaired cognitive function in AD and adult-onset dementia
disorders. Treatments to counteract amyloid-accumulation,
tau hyperphosphorylation, and immunotherapy have been
recommended but failed to produce efects and were there-
fore discontinued in phase II or III clinical trials. At present,
enhancing cholinergic neurotransmission still represents the
main approach to symptomatic treatment of cognitive and
behavioral symptoms of mild and moderate stages of AD.
In line with this therapeutic strategy, various molecules
like linopirdine, an agent that increases hippocampus ACh
release, muscarinic ACh receptor agonists like xanomeline,
and AChE inhibitor compounds like tacrine and physostig-
mine were used [52–55].
obtained for this comparison are used. The enzymes used
to make this comparison are acetylcholinesterase (AChE)
(pdb ID:4M0E) and α- glycosidase (α-Gly) (pdb ID:1R47).
The parameters obtained from the interaction of synthe-
sized compounds with these enzymes are given in Table 2.
As a result of molecular docking calculations, many
parameters obtained for novel molecules are calculated.
The most important parameter among these calculated
parameters is the Docking score parameter of molecules.
The numerical value of this parameter is a numerical
expression of the interactions between molecules and
enzyme. As a result of calculations, the molecule with the
most negative numerical value of this parameter has higher
biological activity than other molecules. It should be well
known that the more interaction between any molecule
and enzyme, the more the numerical value of this param-
eter decreases [46, 59]. Therefore, the most important fac-
tor afecting the biological activities of molecules is the
interactions between compounds 5a-e, 6a-e, and 7a-e of
enzymes. These interactions have many interactions such
as hydrogen bonds, polar and hydrophobic interactions,
π-π, and halogen [60–66]. These interactions are given in
Figs. 2 and 3.
Inhibitors of this enzyme delay the breakdown of car-
bohydrate molecules in the small intestine and diminish
the postprandial blood glucose excursion; hence, inhibition
of glycosidase enzyme has an important efect on poly-
saccharide metabolism, cellular interaction, glycoprotein
processing, and widening opportunities for the discovery
of new therapeutic factors against diseases like obesity,
viral infection, diabetes, and metastatic cancer [56–58]. For
α-glycosidase, IC50 values of ACR as positive control and
some novel molecules the following order: 6d (192.66 nM,
r2: 0.9912)<5b (193.24 nM, r2: 0.9972)<6c (195.37 nM,
r2: 0.9183)<6b (201.52 nM, r2: 0.9715)<ACR (513.41 nM,
r2: 0.9491). Indeed, AG as a glycosidase located in the brush
border of the small intestine can selectively hydrolyze ter-
minal (1→4)-linked α-glucose residues (disaccharides or
starch) to release a single α-glucose molecule. Finally, for
the α-glycosidase, compounds 5a-e, 6a-e, and 7a-e showed
Kis between 176.27 22.87–621.77 69.98 nM (Table 1
and Fig. 1). The results demonstrated that compound 6d
had efective α-glycosidase inhibition efects than that of
acarbose (Ki: 548.13 62.38 nM) as standard α-glycosidase
inhibitor. Also, highly efective Kis were calculated for com-
pound 6d (Ki: 176.27 22.87 nM).
Many more parameters were obtained from the inter-
action of compounds 5a-e, 6a-e, and 7a-e with enzymes.
These parameters explain the interaction between mole-
cule and enzyme from diferent angles. Glide hbond, Glide
evdw, and Glide ecoul parameters of novel compounds
provide information about the chemical interactions
between molecules and enzymes [46, 59]. These interac-
tions are hydrogen bonding, van der Waals, and Coulomb
interactions. On the other hand, Glide emodel, Glide
energy, Glide einternal, and Glide posenum parameters of
novel Schif bases provide information about the interac-
tion pose that occurs in the interaction between molecules
and enzymes [67, 68]. Another parameter obtained from
molecular docking calculations is Glide Ligand Efciency,
which is a numerical value of the efciency of novel Schif
bases against enzymes [69, 70].
After the studied molecules interact with cancer pro-
teins, ADME/T analysis was performed to use the mol-
ecules as advanced drugs. With this analysis, the behavior
of molecules in human metabolism is tried to be predicted
theoretically. With this analysis, how it is absorbed, func-
tioning and excretion process by human tissues and organs
is examined. Among the parameters to be examined for
this analysis, the two most important parameters are Rule-
OfFive [71, 72] and RuleOfThree [73]. These parameters
consist of a combination of many parameters. The numeri-
cal values of these parameters are required to be minimum
zero and maximum three for RuleOfThree and four for
RuleOfFive (Table 3).
Molecular docking
The numerical values of many parameters obtained by
the Schrödinger program used to calculate the biological
activities of compounds 5a-e, 6a-e, 7a-e against enzymes
are compared with the biological activity values of the
molecules [80]. The numerical values of these parameters
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Journal of the Iranian Chemical Society
Table 2 Numerical values of the docking parameters of molecule against enzymes
AChE Docking score Glide
ligand
Glide hbond Glide evdw Glide ecoul Glide emodel Glide energy Glide einternal Glide posenum
efciency
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
−7.84
−0.26
−0.13
−29.94
−10.73
−58.21
−40.68
8.63
11.79
10.06
14.69
20.99
7.48
7.89
–
8.64
–
–
11.73
–
183
394
71
377
330
60
221
–
73
–
–
116
–
−7.66
−8.24
−8.24
−8.77
−8.03
−4.78
–
−8.64
–
–
−0.27
−0.32
−0.25
−0.26
−0.22
−0.14
–
−0.34
–
–
−0.46
−0.40
0.00
−0.36
0.00
−0.30
–
−0.32
–
–
−37.55
−35.03
−32.92
−44.94
−37.76
−44.16
–
−35.14
–
–
−13.55
−14.26
−19.06
−15.27
−16.62
−9.09
–
−11.38
–
–
−77.20
−80.78
−78.60
−86.88
−82.73
−63.13
–
−84.47
–
–
−51.11
−49.29
−51.97
−60.21
−54.39
−53.25
–
−46.52
–
–
6.71
–
−0.23
–
−0.58
–
−40.46
–
−8.24
–
−70.77
–
−48.69
–
α-Gly Docking score Glide
ligand
Glide hbond Glide evdw Glide ecoul Glide emodel Glide energy Glide einternal Glide posenum
efciency
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
7b
7c
7d
7e
−4.29
−4.59
−3.55
−2.96
−2.97
−4.58
−4.38
–
−4.94
−2.88
−2.00
−4.50
–
−0.20
−0.01
−0.47
−0.02
−0.28
−0.06
0.00
−0.16
–
−0.20
0.00
−0.21
−0.16
–
−0.22
–
−21.59
−28.61
−31.36
−26.30
−26.50
−27.28
−33.05
–
−34.88
−31.93
−25.05
−30.80
–
−16.00
−12.00
−6.13
−7.83
−5.91
−14.04
−10.90
–
−10.80
−5.71
−4.40
−11.00
–
−55.62
−51.08
−43.31
−35.60
−35.83
−53.20
−54.68
–
−55.42
−41.33
−35.95
−55.72
–
−37.59
−40.61
−37.49
−34.13
−32.41
−41.32
−43.95
–
−45.68
−37.64
−29.45
−41.81
–
4.73
5.03
7.18
8.30
8.02
11.34
13.88
–
7.28
13.25
2.03
5.87
–
365
334
84
233
109
353
245
–
397
208
5
318
–
−0.16
−0.12
−0.11
−0.11
−0.14
−0.13
–
−0.16
−0.09
−0.07
−0.16
–
−2.30
–
−0.08
–
−25.85
–
−7.93
–
−36.50
–
−33.78
–
5.49
–
371
–
that 6b is more efective and active than other molecules
with a docking score parameter value of − 8.77 against
AChE enzyme and 6d is more efective and active than
other molecules with a docking score parameter value
of − 4.94 against α-Gly enzyme. The presence of more
electron pairs on heteroatoms in molecules and the inter-
action of the regions with higher electron density with
enzymes increase the activity. After this examination,
numerical values of parameters of new molecules can be
used in future in vivo and in vitro studies for the discovery
of new drug candidates.
Conclusion
Biological activity values of compounds 5a-e, 6a-e, and
7a-e against acetylcholinesterase (AChE) and α- glycosi-
dase (α-Gly) were calculated. This novel Schif bases
derived from phenolic Mannich bases were recorded to
have anticholinergic and antidiabetic properties; also,
they are appropriate for future signifcant drug searches.
Afterward, after examining the interaction of these com-
pounds against enzymes, ADME/T analysis was made and
the properties of drugs were examined. As a result of the
docking calculations of the molecules, it has been seen
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Journal of the Iranian Chemical Society
Fig. 2 Presentation interactions of molecule 6b with AChE enzyme
Fig. 3 Presentation interactions of molecule 6d with α-Gly enzyme
recorded on Bruker (100 MHz) spectrometer. Chemical
shifts are reported as δ in ppm relative to tetramethylsilane
(TMS) (δ 0.00 singlets) in deuterated dimethylsulfoxide
(DMSO-d6) and deuterated chloroform (CDCl3). HRMS
spectra were recorded on Agilent 6530 Accurate-Mass
spectrometer, and acetonitrile was used as the solvent.
Experimental
Chemistry
The chemicals used in this study were supplied from
Sigma-Aldrich (Germany). Melting points were deter-
mined on WRS-2A Microprocessor Melting-point
Apparatus and are uncorrected. IR spectra of com-
pounds were recorded using ALPHA-P BRUKER FT-IR
Spectrophotometer.1H-NMR spectra were recorded on
Bruker (400 MHz) spectrometer. 13C-NMR spectra were
General procedure for synthesis compounds 1–3
To a solution of paraformaldehyde (15 mmol) in ethanol
(20 mL), secondary amine (12 mmol) was added and the
mixture was refuxed for an hour. 4-Hydroxy-3-methoxy
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Journal of the Iranian Chemical Society
Table 3 ADME properties of molecule
5a
5b
5c
5d
5e
6a
6b
6c
Referance range
mol_MW
dipole (D)
SASA
FOSA
FISA
293
6.5
577
336
178
64
0.0
987
2
385
5.1
691
310
185
197
0.0
414
12.3
717
310
231
177
0.0
1295
2
385
5.3
733
318
143
271
0.0
375
8.1
445
6.8
794
258
115
422
0.0
1451
2
461
5.0
808
258
170
380
0.0
490
12.6
834
258
215
361
0.0
1528
2
130–725
1.0–12.5
300–1000
0–750
7–330
0–450
0–175
500–2000
0–6
668
310
138
175
44.8
1205
2
PISA
WPSA
volume (A3)
donorHB
1244
3
1268
2
1476
3
accptHB
7.7
0.8
29.4
9.5
17.0
11.7
0.6
−1.9
−1.6
−5.3
51
−1.2
22
−6.0
9.5
0.2
5
8.45
0.8
39.7
13.2
22.3
14.7
1.8
−3.1
−3.4
−6.3
44
−1.5
19
8.7
0.8
41.6
13.6
23.1
13.7
1.8
−3.5
−3.9
−6.3
16
−2.0
6
−6.3
9.7
2.3
6
8.2
0.8
7.7
0.8
7
7.75
0.8
50.8
16.6
25.9
15.4
3.7
−5.2
−5.5
−7.7
60
−1.5
26
8
2.0–20.0
0.75–0.95
13.0–70.0
4.0–18.0
8.0–35.0
4.0–45.0
−2.0–6.5
−6.5–0.5
−6.5–0.5
*
**
−3.0–1.2
**
Kp in cm/hr
7.9–10.5
−0.9–1.7
1–8
glob (Sphere=1)
QPpolrz (A3)
QPlogPC16
QPlogPoct
QPlogPw
QPlogPo/w
QPlogS
CIQPlogS
QPlogHERG
QPPCaco (nm/sec)
QPlogBB
QPPMDCK (nm/sec)
QPlogKp
0.8
50.8
16.1
24.1
13.3
4.5
−5.5
−5.5
−7.8
202
−0.8
97
0.8
52.7
17.1
26.6
14.5
3.8
−5.7
−6.0
−7.7
22
−2.0
9
−5.4
9.1
2.2
5
42.1
13.5
21.3
13.5
2.5
−4.1
−3.6
−7.2
109
−1.1
50
38.6
12.4
20.5
12.2
2.4
−3.3
−3.5
−6.0
122
−0.8
99
−5.4
9.4
0.1
−4.5
9.5
0.1
−4.7
9.4
0.6
−3.4
9.0
0.1
4
−4.5
9.0
0.0
IP (ev)
EA (eV)
#metab
6
5
6
5
QPlogKhsa
Human Oral Absor
Per. Human Oral Absor
PSA
RuleOfFive
RuleOfThree
Jm
−0.4
3
61
106
0
0
−0.1
2
67
116
0
0
0.0
2
59
140
0
1
0.1
3
78
104
0
0
0.0
0.1
3
78
92
0
0
0.0
0.8
3
94
89
0
0
0.0
0.6
3
81
112
0
0
0.0
0.7
2
73
136
0
2
−1.5–1.5
–
***
7–200
Maximum is 4
Maximum is 3
–
0.0
0.0
0.0
0.0
6d
6e
7a
7b
7c
7d
7e
Referance range
mol_MW
dipole (D)
SASA
FOSA
FISA
PISA
WPSA
461
5.2
848
266
129
453
0.0
1501
2
7.5
451
8.0
381
7.6
713
365
108
240
0.0
1292
2
6
397
5.9
727
365
164
197
0.0
1316
3
6.75
0.8
426
13.4
753
365
209
178
0.0
1368
2
7
397
6.1
767
373
122
271
0.0
1341
2
6.5
387
8.9
130–725
1.0–12.5
300–1000
0–750
7–330
0–450
0–175
500–2000
0–6
2.0–20.0
0.75–0.95
13.0–70.0
4.0–18.0
8.0–35.0
4.0–45.0
783
258
123
357
44.8
1438
2
7
0.8
49.7
15.8
24.0
12.9
702
365
117
175
44.8
1278
2
6
0.8
41.5
12.9
20.6
10.5
volume (A3)
donorHB
accptHB
glob (Sphere=1)
QPpolrz (A3)
QPlogPC16
QPlogPoct
QPlogPw
0.7
0.8
0.8
0.8
53.1
17.1
24.9
14.3
42.7
13.2
20.6
10.9
42.6
13.7
22.3
13.0
44.5
14.2
23.3
12.0
45.0
14.1
21.4
11.8
1 3

Journal of the Iranian Chemical Society
Table 3 (continued)
6d
6e
7a
7b
7c
7d
7e
Referance range
QPlogPo/w
QPlogS
CIQPlogS
QPlogHERG
QPPCaco (nm/sec)
QPlogBB
QPPMDCK (nm/sec)
QPlogKp
4.5
−6.3
−5.7
−8.5
149
−1.1
70
−3.6
9.0
0.0
4
4.4
−5.5
−5.6
−7.4
167
−0.7
139
−3.8
9.0
0.6
5
3.7
−4.5
−4.2
−6.6
233
−0.6
113
−4.0
9.4
0.0
4
3.0
−4.2
−4.2
−6.4
69
−1.3
30
−5.0
9.4
0.0
5
3.0
−4.6
−4.7
−6.5
26
−1.8
10
−5.9
9.6
2.2
5
3.8
−5.2
−4.4
−7.3
172
−0.9
81
−4.1
9.4
0.0
4
3.8
−4.4
−4.3
−6.1
192
−0.6
162
−4.4
9.4
0.5
5
−2.0–6.5
−6.5–0.5
−6.5–0.5
*
**
−3.0–1.2
**
Kp in cm/hr
7.9–10.5
−0.9–1.7
1–8
IP (ev)
EA (eV)
#metab
QPlogKhsa
Human Oral Absor
Per. Human Oral Absor
PSA
RuleOfFive
RuleOfThree
Jm
0.8
1
92
100
0
1
0.8
3
93
87
0
0
0.0
0.6
3
91
83
0
0
0.0
0.4
3
77
106
0
0
0.5
2
70
130
0
0
0.6
3
89
93
0
0
0.0
0.5
3
90
81
0
0
0.0
−1.5–1.5
–
***
7–200
Maximum is 4
Maximum is 3
–
0.0
0.0
0.0
*Corcern below−5, **a<25 is poor and a>500 is great, ***b<25 is poor and b>80 is high
benzaldehyde (vanillin) (10 mmol) was added to this mix-
ture and refuxed for 4 h. Reaction progress was monitored
by TLC (hexane: ethyl acetate–7:3). After completion, half
of the solvent was removed under reduced pressure and the
mixture was left in the freezer overnight and the formed
solid was fltered of. The crude product was recrystallized
from ethanol (Fig. 4).
868, 705; 1H-NMR (400 MHz, CDCl3) δ 9.79 (s, 1H), 7.36
(m, 1H), 7.19 (m, 1H) 3.95 (s, 3H), 3.82 (s, 2H), 3.78 (m,
4H), 2.63 (m, 4H); 13C-NMR (100 MHz, CDCl3)δ 190.6,
153.5, 148.6, 128.5, 125.5, 120.3, 109.8, 66.6 (2C), 61.1,
56.0, 52.7 (2C).
4‑Hydroxy‑3‑methoxy‑5‑((4‑phenylpiperazin‑1‑yl)methyl)
benzaldehyde (2)
4‑Hydroxy‑3‑methoxy‑5‑(morpholinomethyl)benzaldehyde
(1)
White solid; yield: 90%, mp: 156 °C (lit: 156–157 °C) [75].
IR (ATR, cm−1) νmax2959, 2938, 2827, 2737, 1677, 1586,
1315, 1235, 1141, 760, 691;1H-NMR (400 MHz, CDCl3)
δ10.90 (brs, 1H), 9.81 (s, 1H), 7.39–7.38 (m, 1H), 7.32–7.27
White solid; yield: 85%, mp: 99–100 °C [74]. IR (ATR,
cm−1) νmax2945, 2866, 2829, 2733, 1647, 1592, 1270, 1120,
Fig. 4 Synthesis of compounds
O
O
1–3
O
H
N
+
+
R
R
H
H
O
EtOH, ref. 5 h.
O
N
OH
R
R
OH
1-3
R2 NH :
HN
O
HN
N
HN
1
2
3
1 3

Journal of the Iranian Chemical Society
(m, 2H), 7.22–7.17 (m, 1H) 6.95–6.86 (m, 3H), 3.96 (s,
3H), 3.89 (s, 2H), 3.28 (m, 4H), 2.80 (m,4H); 13C-NMR
(100 MHz, CDCl3)δ 190.6, 153.7, 150.7, 148.7, 129.2 (2C),
128.4, 125.5, 120.6, 120.4, 116.5 (2C), 109.9, 60.8, 56.0,
52.5 (2C), 49.2 (2C).
was removed under reduced pressure and the mixture was
left in the freezer overnight and the formed solid was fl-
tered of. The crude product was recrystallized from ethanol
(Fig. 6).
N’‑(4‑hydroxy‑3‑methoxy‑5‑(morpholinomethyl)
benzylidene)benzohydrazide(5a)
4‑Hydroxy‑3‑methoxy‑5‑((3‑methylpiperidin‑1‑yl)methyl)
benzaldehyde (3)
White solid, yield: 84%, mp:231–233 °C, IR (ATR, cm−1)
νmax3183, 3062, 2951, 1639, 1601, 1303, 1116, 858, 690;1H-
NMR (400 MHz, CDCl3, δ/ppm): δ 10.20 (s, 1H), 8.30 (s,
1H), 7.92 (d, 2H, J=8.0 Hz), 7.50 (t, 1H, J=7.2 Hz),7.40 (t,
1H, J=7.4 Hz), 7.24 (s, 1H), 6.91 (s, 1H), 3,81 (s, 3H), 3.72
(m, 4H), 3.62 (s, 2H), 2,52 (m, 4H). 13C-NMR (100 MHz,
CDCl3,δ/ppm): δ164.5, 149.5, 149.2, 148.2, 133.2, 131.9,
128.6 (2C), 127.6 (2C), 125.0, 121.9, 120.4, 109.1, 66.6
(2C), 61.0, 55.9, 52.7 (2C). HRMS (Q-TOF) m/z calcd for
C20H23N3O4: 369.1689, found: 370.1745 [M+H]+.
Light brown solid; yield: 88%, mp: 142–144 °C; IR (ATR,
cm−1) νmax2946, 2922, 2853, 2748, 1651, 1592, 1271, 1147,
864, 707;1H-NMR (400 MHz, CDCl3) δ11.55 (s, 1H), 9.76
(s, 1H), 7.33 (m, 1H), 7.15 (m, 1H) 3.94 (s, 3H), 3.78 (m,
2H), 2.96–2.90 (m, 2H), 2.11 (t, J=10.5 Hz, 1H), 1.83–1.58
(m, 5H), 0.98–0.95 (m, 1H), 0.89 (d, J =6.3 Hz, 3H),13C-
NMR (100 MHz, CDCl3)δ 190.6, 154.8, 148.6, 127.8, 125.4,
120.8, 109.5, 61.2, 60.7, 55.9, 53.2, 32.2, 31.0, 25.0, 19.2.
General procedure for synthesis compounds 4a‑e
N’‑(4‑hydroxy‑3‑methoxy‑5‑(morpholinomethyl)benzylide
ne)‑4‑hydroxybenzohydrazide(5b)
Concentrated H2SO4 (1 mL) was added to the solution of
suitable carboxylic acids (10 mmol) in methanol (20 mL)
and refuxed for 2 h. The mixture was cooled to room tem-
perature, and saturated NaHCO3 solution (50 mL) was added
slowly. The aqueous layer was extracted with dichlorometh-
ane (3×15 mL). Organic layer was dried with sodium sulfate
and the solvent was removed by evaporation under reduced
pressure. The crude product was dissolved ethanol (20 ml)
and hydrazinium hydroxide (5 ml, 80%) was added to this
mixture. It was refuxed for 4 h and left in the freezer over-
night. Formed crystals were fltered of, dried and recrystal-
lized from ethanol (Fig. 5).
White solid, yield: 88%, mp:249–250 °C, IR (ATR, cm−1)
νmax3193, 3069, 2937, 1649, 1607, 1304, 1108, 852, 698;1H-
NMR (400 MHz, DMSO-d6, δ/ppm): δ 11.51 (s, 1H), 8.31
(s, 1H), 7.80 (d, 2H, J=8.0 Hz), 7.22 (s, 1H), 7.07 (s, 1H),
6.86 (d, 2H, J=8.0 Hz), 3,83 (s, 3H), 3.63 (s, 2H), 3.61 (m,
4H), 2.45 (m, 4H). 13C-NMR (100 MHz, DMSO-d6, δ/ppm):
δ162.6, 160.5, 147.9, 147.7, 147.4, 129.6 (2C), 125.1, 124.0,
122.5 (2C), 121.9, 115.0, 108.1, 66.1 (2C), 58.2, 55.6, 52.7
(2C). HRMS (Q-TOF) m/z calcd for C20H23N3O5: 385.1638,
found: 386.1695 [M+H]+.
General procedure for synthesis compounds 5a‑e, 6a‑e,
and 7a‑e
N’‑(4‑hydroxy‑3‑methoxy‑5‑(morpholinomethyl)benzylide
ne)‑3‑nitrobenzohydrazide(5c)
Suitable aldehyde (1–3) (10 mmol) and hydrazide (4a-e)
(10 mmol) were dissolved in absolute ethanol (20 mL) and
4–5 drops of acetic acid was added. Reaction mixture was
refuxed for 2–3 h. Compounds 5a-e and 6a-e precipitated
during the reaction. For compounds 7a-e, half of the solvent
Yellow solid, yield: 90%, mp:235–237 °C [76]. IR (ATR,
cm−1) νmax3176, 3080, 2945, 1644, 1611, 1532, 1348,
1
1308, 1115, 858, 703; H-NMR (400 MHz, DMSO-
d6, δ/ppm): δ 12.04 (s, 1H), 8.75 (s, 1H), 8.43 (d, 1H,
J = 8.0 Hz), 8.36 (s, 1H), 7.84 (t, 1H, J = 8.0 Hz), 7.26
Fig. 5 Synthesis of compounds
O
O
N2H4.H2O
O
4a-e
NH2
CH3OH, Con. H2SO4
ref. 2 h.
EtOH, ref. 4 h.
R
OH
R
N
H
R
O
4a-e
O
O2N
S
R:
HO
a
c
e
b
d
1 3

Journal of the Iranian Chemical Society
O
O
O
N
O
O
N
O
N
H
R
R
N
H
N
O
OH
N
OH
O
NH2
R
N
H
O
O
EtOH, AcOH (cat.)
ref. 2 h.
EtOH, AcOH (cat.)
ref. 2 h.
N
OH
N
O
OH
4a-e
O
5a-e
7a-e
O
EtOH, AcOH (cat.)
ref. 2 h.
N
N
OH
O
N
N
H
R
N
N
O
OH
6a-e
Fig. 6 Synthesis of compounds 5a-e, 6a-e, and 7a-e
(s, 1H), 7.12 (s, 1H), 3,85 (s, 3H), 3.65 (s, 2H), 3.61 (m,
4H), 2.46 (m, 4H).:13C-NMR (100 MHz, DMSO-d6, δ/
ppm): δ160.7, 149.3, 148.4, 147.8, 147.7, 134.9, 134.0,
130.2, 126.2, 124.6, 122.5, 122.3, 122.2, 108.2, 66.1
(2C), 58.2, 55.6, 52.7 (2C). HRMS (Q-TOF) m/z calcd for
C20H22N4O6: 414.1539, found: 415.1599 [M + H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑(morpholinomethyl)
benzylidene)thiophene‑2‑carbohydrazide(5e)
Beige solid, yield: 87%, mp:217–219 °C, IR (ATR, cm−1)
νmax3169, 3043, 2945, 1626, 1584, 1300, 1115, 838, 711;1H-
NMR (400 MHz, CDCl3,δ/ppm): δ 10.87 (s, 1H), 8.26 (s,
1H), 7.93 (s, 1H), 7.71 (d, 1H, J=4.0 Hz), 7.43 (s, 1H), 7.19
(s, 1H), 6.98 (s, 1H), 3,98 (s, 3H), 3.78 (m, 6H), 2.62 (m,
4H). 13C-NMR (100 MHz, CDCl3,δ/ppm): δ163.1, 149.4,
148.4, 145.2, 135.5, 134.5, 132.7, 126.5, 125.3, 121.9,
120.6, 109.2, 66.7 (2C), 61.4, 55.9, 52.8 (2C). HRMS
(Q-TOF) m/z calcd for C18H21N3O4S: 375.1253, found:
376.1305 [M+H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑(morpholinomethyl)benzylide
ne)‑2‑phenoxyacetohydrazide(5d)
White solid, yield: 89%, mp:205–206 °C, IR (ATR, cm−1)
νmax3183, 3036, 2951, 1661, 1591, 1298, 1114, 832, 690;1H-
NMR (400 MHz, DMSO-d6, δ/ppm): δ 11.48 and 11.44 (2 s,
1H), 8.20 and 7.90 (2 s, 1H), 7.34–7.27 (m, 2H), 7.21 (d, 1H,
J=8.0 Hz), 7.07 (s, 1H), 7.00 (d, 1H, J=8.0 Hz), 6.96–6.91
(m, 2H), 5.14 and 4.65 (2 s, 2H),3,81 (s, 3H), 3.63 (s, 2H),
3.60 (m, 4H), 2.44 (m, 4H). 13C-NMR (100 MHz, DMSO-
d6, δ/ppm): δ168.8, 163.9, 158.2, 157.7, 148.3, 147.9, 144.2,
129.5, 129.4, 124.6, 122.1, 121.4, 120.6, 114.6, 108.4, 66.1
(2C), 64.6, 58.3, 55.6, 52.6 (2C). HRMS (Q-TOF) m/z calcd
for C21H25N3O5: 399.1794, found: 400.1846 [M+H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑((4‑phenylpiperazin‑1‑yl)
methyl)benzylidene)benzohydrazide(6a)
White solid, yield: 93%, mp:249–251 °C, IR (ATR, cm−1)
νmax3198, 3057, 2949, 1649, 1594, 1312, 1110, 686;1H-
NMR (400 MHz, DMSO-d6, δ/ppm): δ 11.74 (s, 1H),
8.37 (s, 1H), 7.92 (d, 2H, J=8.0 Hz), 7.59–7.53 (m, 3H),
7.27–7.20 (m, 3H), 7.12 (s, 1H), 6.94 (d, 2H, J =8.0 Hz),
1 3

Journal of the Iranian Chemical Society
6.79 (t, 1H, J=7.2 Hz) 3,85 (s, 3H), 3.72 (s, 2H), 3.17 (m,
4H), 2,62 (m, 4H). 13C-NMR (100 MHz, DMSO-d6, δ/ppm):
δ162.9, 150.8, 148.3, 147.8, 133.6, 131.6, 128.9 (3C), 128.4
(2C), 127.5 (2C), 124.9, 122.7, 122.0, 119.0, 115.5 (2C),
108.2, 57.9, 55.6, 52.1 (2C), 48.2 (2C). HRMS (Q-TOF) m/z
calcd for C26H28N4O3: 444.2161, found: 445.2220 [M+H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑((4‑phenylpiperazin‑1‑yl)
methyl)benzylidene)thiophene‑2‑carbohydrazide(6e)
Beige solid, yield: 92%, mp:222–224 °C, IR (ATR, cm−1)
νmax3216,3187, 3061, 2946, 1627, 1583, 1298, 842, 713;1H-
NMR (400 MHz, CDCl3, δ/ppm): δ 10.44 (s, 1H), 8.25 (s,
1H), 7.89 (s, 1H), 7.70 (s, 1H), 7.44 (s, 1H), 7.30–7.25 (m,
2H), 7.19 (s, 1H), 6.99 (s, 1H), 6.91–6.88 (m, 3H), 3,98 (s,
3H), 3.84 (s, 2H), 3.26 (m, 4H), 2,78 (m, 4H). 13C-NMR
(100 MHz, CDCl3, δ/ppm): δ162.9, 159.7, 150.8, 149.6,
148.5, 145.0, 135.5, 134.5, 129.3 (2C), 126.5, 125.1, 121.9,
120.9, 120.4, 116.5 (2C), 109.2, 61.0, 56.0. 52.5 (2C),
49.1 (2C). HRMS (Q-TOF) m/z calcd for C24H26N4O3S:
450.1726, found: 451.1794 [M+H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑((4‑phenylpiperazin‑1‑yl)
methyl)‑4‑hydroxybenzylidene)benzohydrazide(6b)
White solid, yield: 91%, mp:254–256 °C, IR (ATR, cm−1)
νmax3273, 3061, 2939, 1646, 1598, 1308, 1114, 756;1H-
NMR (400 MHz, DMSO-d6, δ/ppm): δ 11.52 (s, 1H), 8.33
(s, 1H), 7.81 (d, 2H, J=8.2 Hz), 7.27–7.20 (m, 3H), 7.09 (s,
1H), 6.94 (d, 2H, J=8.0 Hz), 6.79 (t, 1H, J=7.2 Hz), 3,84
(s, 3H), 3.71 (s, 2H), 3.16 (m, 4H), 2,62 (m, 4H). 13C-NMR
(100 MHz, DMSO-d6, δ/ppm): δ162.5, 160.5, 150.8, 148.1,
147.7, 147.4, 129.6, 128.9 (3C), 125.1, 124.0, 122.6, 121.8,
119.0, 115.5 (3C), 114.9, 108.1, 58.0, 55.6, 52.1 (2C), 48.2
(2C). HRMS (Q-TOF) m/z calcd for C26H28N4O4: 460.2111,
found: 461.2167 [M+H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑((3‑methylpiperidin‑1‑yl)
methyl)benzylidene)benzohydrazide(7a)
Beige solid, yield: 79%, mp:199–201 °C, IR (ATR, cm−1)
νmax3230, 3066, 2929, 1643, 1603, 1298, 692;1H-NMR
(400 MHz, DMSO-d6, δ/ppm): δ 11.79 (s, 1H), 8.37 (s,
1H), 7.94 (d, 2H, J=8.0 Hz), 7.58–7.52 (m, 4H), 7.25 (s,
1H), 7.01 (s, 1H), 3,81 (s, 3H), 3.65 (s, 2H), 2.78 (m, 2H),
1.97 (t, 1H, J=10.40 Hz), 1.70–1.43 (m, 5H), 0,89–0,80 (m,
4H). 13C-NMR (100 MHz, DMSO-d6, δ/ppm):δ162.9, 149.2,
148.4, 147.7 133.6, 131.6, 128.4 (2C), 127.5 (2C), 124.7,
122.2, 121.5, 108.2, 60.2, 59.6, 55.5, 52.6, 32.0, 30.6, 24.8,
19.2. HRMS (Q-TOF) m/z calcd for C22H27N3O3: 381.2052,
found: 382.2111 [M+H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑((4‑phenylpiperazin‑1‑yl)
methyl)benzylidene)‑3‑nitrobenzohydrazide(6c)
Yellow solid, yield: 93%, mp:223–224 °C [76]. IR (ATR,
cm−1) νmax3194, 3075, 2946, 1641, 1599, 1531, 1348, 1306,
688;1H-NMR (400 MHz, DMSO-d6, δ/ppm): δ 12.04 (s,
1H), 8.75 (s, 1H), 8.44 (d, 1H, J = 7.8 Hz), 8.37 (d, 2H,
J = 9.8 Hz),7.84 (t, 1H, J = 8.0 Hz),7.28–7.15 (m, 4H),
6.94 (d, 2H, J = 8.0 Hz), 6.79 (t, 1H, J = 7.2 Hz), 3,86 (s,
3H), 3.73 (s, 2H), 3.17 (m, 4H), 2,63 (m, 4H). 13C-NMR
(100 MHz, DMSO-d6, δ/ppm): δ160.7, 150.8, 149.3, 148.5,
147.8, 134.9, 134.1, 130.3, 128.9 (3C), 126.2, 124.7, 122.7,
122.2, 119.0, 115.5 (3C), 108.3, 57.9, 55.7, 52.1 (2C), 48.2
(2C). HRMS (Q-TOF) m/z calcd for C26H27N5O5: 489.2012,
found: 490.2080 [M+H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑((3‑methylpiperidin‑1‑yl)
methyl)benzylidene)‑4‑hydroxybenzohydrazide(7b)
Beige solid, yield: 81%, mp:180–182 °C, IR (ATR, cm−1)
νmax3177, 3010, 2932, 1641, 1600, 1276, 1169, 848;1H-
NMR (400 MHz, CH3OD, δ/ppm): δ 8.20 (s, 1H), 7.84
(d, 2H, J=8.6 Hz), 7.60 (s, 1H), 7.02 (s, 1H), 6.89 (d, 2H,
J=8.6 Hz), 3,91 (s, 3H), 3.85 (s, 2H), 3.06–3.00 (m, 2H),
2.30 (t, 1H, J=11.2 Hz), 2.05–1.99 (m, 1H), 1.79–1.59 (m,
4H), 1.04–0.98 (m, 1H), 0,91 (d, 3H, J=6.4 Hz).13C-NMR
(100 MHz, CH3OD, δ/ppm): δ166.8, 162.8, 151.9, 150.2,
149.9, 130.8 (2C), 126.2, 124.8, 124.6, 121.3 (2C), 116.4,
110.1, 61.2, 60.4, 57.5, 53.9, 32.9, 31.9, 25.6, 19.6. HRMS
(Q-TOF) m/z calcd for C22H27N3O4: 397.2002, found:
398.2066 [M+H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑((4‑phenylpiperazin‑1‑yl)
methyl)benzylidene)‑2‑phenoxyacetohydrazide(6d)
White solid, yield: 90%, mp:210–212 °C, IR (ATR, cm−1)
νmax3174, 3035, 2973, 1657, 1593, 1314, 1226, 749,
686;1H-NMR (400 MHz, DMSO-d6, δ/ppm): δ 11.50 and
11.46 (2 s, 1H), 8.23 and 7.92 (2 s, 1H), 7.34–7.19 (m,
5H), 7.10 (s, 1H), 7.00 (d, 1H, J= 8.2 Hz), 6.93 (m, 4H),
6.79 (t, 1H, J = 7.0 Hz), 5.14 and 4.65 (2 s, 2H), 3,82 (s,
3H), 3.70 (s, 2H), 3.15 (m, 4H), 2.61 (m, 4H). 13C-NMR
(100 MHz, DMSO-d6, δ/ppm): δ168.8, 163.9, 158.2, 157.7,
150.8, 148.3, 147.7, 144.2, 129.5, 128.9, 124.6, 122.6,
121.2, 120.6, 119.0, 115.5, 114.6, 108.5, 66.4, 64.6, 57.9,
55.6, 52.1 (2C), 48.2 (2C). HRMS (Q-TOF) m/z calcd for
C27H30N4O4: 474.2267, found: 475.2339 [M+H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑((3‑methylpiperidin‑1‑yl)
methyl)benzylidene)‑3‑nitrobenzohydrazide (7c)
Beige solid, yield: 80%, mp:230–231 °C, IR (ATR, cm−1)
νmax3241, 3084, 2943, 1642, 1585, 1528, 1349, 1301,
765;1H-NMR (400 MHz, DMSO-d6, δ/ppm): δ 12.05 (brs,
1H), 8.75 (s, 1H), 8.43 (d, 2H, J = 7.8 Hz), 8.36 (d, 1H,
1 3

Journal of the Iranian Chemical Society
J=6.8 Hz), 7.83 (t, 1H, J=7.8 Hz),7.24 (s, 1H),7.03 (s, 1H),
3.82 (s, 3H), 3.67 (s, 2H), 2.80 (m, 2H), 2.02–1.97 (m, 1H),
1.73–1.45 (m, 5H), 0.90–0.82 (m, 4H). 13C-NMR (100 MHz,
DMSO-d6, δ/ppm):δ160.7, 149.4, 149.3, 147.7, 134.9, 134.0
(2C), 130.2, 126.1, 124.4, 122.3, 122.2, 121.7, 108.3, 60.2,
59.5, 55.5, 52.6, 32.0, 30.7, 24.8, 19.2. HRMS (Q-TOF) m/z
calcd for C22H26N4O5: 426.1903, found: 427.1971 [M+H]+.
enzyme activity was determined using p-NPG as substrate
according to the procedure of Tao et al.; [82] Samples were
prepared by dissolving 20 mg in 2 mL (EtOH:H2O). If all
enzyme inhibition was achieved, multiple solutions were
prepared in phosphate bufer. First, 75 µL of phosphate
bufer was mixed with phosphate bufer (0.15 U/mL, pH
7.4) and 20 µL of enzyme solution in 5 µL of sample. It was
then pre-incubated at 35 °C for 10 min before adding p-NPG
to the start of the reaction. Also, after pre-incubation, 20 µL
of p-NPG in phosphate bufer (5 mM, pH=7.4) was added
and incubation was carried out again at 35 °C. IC50 and Ki
values were calculated by curve ftting the data. Acarbose
compound was used as a positive control. Absorbances were
measured spectrophotometrically at 405 nm [83–85]. One
unit of α-glycosidase is the amount of enzyme (pH: 7.4) that
catalyzes 1.0 mol of substrate hydrolysis per minute [86].
The inhibitory efect of novel Schif bases from Phenolic
Mannich bases (5a-e, 6a-e, and 7a-e) on AChE activity was
performed according to spectrophotometric method of Ell-
man [77] as described previously [78–81]. α-Glycosidase
inhibition efect of novel compounds (5a-e, 6a-e, and 7a-e)
was evaluated according to the method of Tao et al. [82] The
absorbances of samples were recorded at 405 nm as previ-
ously described [83–86].
N’‑(4‑hydroxy‑3‑methoxy‑5‑((3‑methylpiperidin‑1‑yl)
methyl)benzylidene)‑2‑phenoxyacetohydrazide(7d)
Beige solid, yield: 82%, mp:172–173 °C, IR (ATR, cm−1)
νmax3198, 3060, 2925, 167, 1593, 1223, 1119, 745, 686;1H-
NMR (400 MHz, DMSO-d6, δ/ppm): δ 11.47 and 11.46 (2 s,
1H), 8.19 and 7.89 (2 s, 1H), 7.34–7.27 (m, 2H), 7.20 (s,
1H), 7.03–7.91 (m, 4H),5.13 and 4.64 (2 s, 2H), 3.79 (s,
3H), 3.71 (s, 2H), 2.83 (m, 2H), 2.07 (m, 1H), 1.81–1.46 (m,
5H), 0,93–0.91 (m, 1H), 0.84 (d, 3H, J=6.2 Hz). 13C-NMR
(100 MHz, DMSO-d6, δ/ppm):δ168.8, 163.9, 158.2, 157.7,
149.1, 148.9, 148.3, 147.7, 144.2, 129.5, 129.3,124.4, 121.8,
121.2, 121.0, 120.6, 114.6, 114.4, 108.7, 108.5, 66.4, 64.6,
60.0, 55.5, 52.5, 31.8, 30.5, 24.6, 19.2. HRMS (Q-TOF) m/z
calcd for C23H29N3O4: 411.2158, found: 412.2222 [M+H]+.
N’‑(4‑hydroxy‑3‑methoxy‑5‑((3‑methylpiperidin‑1‑yl)
methyl)benzylidene)thiophene‑2‑carbohydrazide(7e)
Docking studies
Beige solid, yield: 80%, mp:185–186 °C, IR (ATR, cm−1)
νmax3225, 3069, 2926, 1633, 1603, 1294, 842, 705;1H-NMR
(400 MHz, DMSO-d6, δ/ppm): δ 11.75 (s, 1H), 8.31 (s, 1H),
8.05–7.86 (m, 2H), 7.36 (s, 1H), 7.22 (m, 2H), 7.06–7.02
(m, 1H), 3,84 (s, 3H), 3.67 (s, 2H), 2.80–2.78 (m, 2H), 2.00
(t, 1H, J=10.8 Hz), 1.76–1.45 (m, 5H), 0.93–0.88 (m, 1H),
0.82 (d, 3H, J=6.4 Hz). 13C-NMR (100 MHz, DMSO-d6,
δ/ppm):δ161.0, 157.5, 149.2, 147.8, 144.2, 134.9, 133.0,
131.5, 128.7, 126.5, 124.6, 121.4, 108.4, 60.2, 59.5, 55.5,
52.6, 32.0, 30.7, 24.8, 19.2. HRMS (Q-TOF) m/z calcd for
C20H25N3O3S: 387.1617, found: 388.1679 [M+H]+.
One of the most important methods used to calculate
the theoretical biological activities of molecules against
enzymes is molecular docking. In the calculations made by
molecular docking method, many parameters are obtained
from the interactions of molecules with enzymes to compare
biological activities. These parameters are very important
parameters to explain the theoretical biological activities
of molecules [45]. AChE and α-Gly were used to calcu-
late the biological activity of Schif bases from Phenolic
Mannich bases (5a-e, 6a-e, 7a-e). In this study, the crystal
structure of acetylcholinesterase (AChE) (pdb ID:4M0E)
and α-glycosidase (α-Gly) (pdb ID:1R47) [87] was used for
docking study of compounds 5a-e, 6a-e, 7a-e. Molecular
docking calculations to calculate the biological activity of
compounds 5a-e, 6a-e, 7a-e were performed using Maestro
Molecular modeling platform (version 12.2) by Schrödinger.
Proteins and new molecules should be prepared for calcula-
tions using the Maestro Molecular modeling platform (ver-
sion 12.2) by Schrödinger program. In docking calculations,
diferent processes are performed for novel molecules and
enzymes at each stage. It was primarily used from Gauss-
ian software program [88] to obtain optimized structures
of novel compounds. Using these optimized structures, all
calculations were made with the Maestro Molecular mod-
eling platform (version 12.2) by Schrödinger, LLC [89].
The Maestro Molecular modeling platform (version 12.2)
Enzyme inhibition studies
The efect of these compounds on the acetylcholinester-
ase enzyme was investigated according to the Ellman [77]
method. For this purpose, IC50 and Ki values were found
and inhibition types were determined. The basis of this
method can be explained as follows: Cholinesterases cata-
lyze the breakdown of acetylcholine into thiocholine and
acetate. It forms yellow colored 5-thio-2-nitrobenzoic acid,
which is formed by the reaction of thiocholine and DTNB,
which is released as a product. The resulting compound
color gave an absorbance at 412 nm. The absorbance of the
sample and blank cuvettes was measured at a wavelength of
412 nm and for 5 min [78–81]. Additionally, α-glycosidase
1 3

Journal of the Iranian Chemical Society
13. B. Lal, V.G. Gund, N.B. Bhise, A.K. Gangopadhyay, Bioorg.
Med. Chem. 12, 1751 (2004)
by Schrödinger comes together from many modules. In the
frst module between these modules, the protein preparation
module [90, 91] is used to prepare the enzymes formed by
proteins for calculations. In the next module, the LigPrep
module [92, 93] was used to prepare compounds 5a-e, 6a-e,
7a-e for calculations. In the next module, The Glide ligand
docking module [94] was used for enzymes to interact with
novel molecules. In all modules used, OPLS3e method
was used in all calculations for docking calculations of
novel compounds and proteins. After docking calculations,
ADME/T analysis (absorption, distribution, metabolism,
excretion, and toxicity) was performed to examine the prop-
erties of novel compounds for future drug use. The Qik-
prop module [95] of the Schrödinger software was used for
ADME/T analysis.
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Authors’ contributions FST, PT, and BT contributed equally to the
curation and writing of manuscript; IHD provided NMR and HRMS
data; KŞ and IG performed conceptualization, reviewing, and editing
of the manuscript.
20. M.D. Aytemir, E. Septio_glu, Ü. Çalis¸ , Arzneimittelforschung
2010, 60, 22.
21. IYu. Chukicheva, I.V. Fedorova, E.V. Buravlev, A.E. Lumpov,
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Funding No funding was received for conducting this study.
Declarations
23. Q. Zhao, X. Liu, L. Zhang, X. Shen, J. Qi, J. Wang, N. Qian, L.
Deng, Calcif. Tissue Int. 93, 172 (2013)
Conflicts of interest The authors declare that they have no conficts of
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interest to disclose.
25. M.I. Fernandez-Bachiller, C. Perez, G.C. Gonzalez-Munoz,
S. Conde, M.G. Lopez, M. Villarroya, A.G. García, M.I. Rod-
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Ethics approval and consent to participate Not applicable. The manu-
script does not contain data collected from humans or animals.
26. B.P. Bandgar, S.A. Patil, R.N. Gacche, B.L. Korbad, B.S. Hote,
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27. A.M. Velazquez, V. Martínez, V. Abrego, M.A. Balboa, L.A. Tor-
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