4,5-dimethoxy-2-nitrobenzohydrazides and 1-(1-benzylpiperidin-4-yl)ethan-1-ones as potential antioxidant/cholinergic endowed small molecule leads

Article abstract of DOI:10.3390/scipharm86010002

The objective of this research is to generate leads for developing our ultimate poly-active molecules with utility in central nervous system (CNS) diseases. Indeed, poly-active molecules capable of mitigating brain free radical damage while enhancing acetylcholine signaling (via cholinesterase inhibition) are still being sought for combating Alzheimer’s disease (AD). We differentiate “poly-active” agents from “multi-target” ones by defining them as single molecular entities designed to target only specific contributory synergistic pharmacologies in a disease. For instance, in AD, free radicals either initiate or act in synergy with other pharmacologies, leading to disease worsening. For this preliminary report, a total of 14 (i.e., 4,5-dimethoxy-2-nitrobenzohydrazide plus 1-(1-benzylpiperidin-4-yl)ethan-1-one) derivatives were synthesized and screened, in silico and in vitro, for their ability to scavenge free radicals and inhibit acetylcholinesterase (AChE)/butyrylcholinesterase (BuChE) enzymes. Overall, six derivatives (4a, 4d, 4e, 4f, 4g, 9b) exhibited potent (>30%) antioxidant properties in the oxygen radical absorbance capacity (ORAC) assay. The antioxidant values were either comparable or more potent than the comparator molecules (ascorbic acid, resveratrol, and trolox). Only three compounds (4d, 9a, 9c) yielded modest AChE/BuChE inhibitions (>10%). Please note that a SciFinder substance data base search confirmed that most of the compounds reported herein are new, except 9a and 9c which are also commercially available.

Full text of DOI:10.3390/scipharm86010002

Scientia
Pharmaceutica
Communication
4,5-Dimethoxy-2-nitrobenzohydrazides and
1-(1-Benzylpiperidin-4-yl)ethan-1-ones as Potential
Antioxidant/Cholinergic Endowed Small
Molecule Leads
Rukhsar Banu 1, Jason Gerding 1, Cynthia Franklin 1, Donald Sikazwe 1,*, William Horton 2,
Marianna Török 2 ID , Julian Davis 3, Kwan H. Cheng 4, Muziya Nakazwe 5
and Bereket Mochona 6
1
Pharmaceutical Sciences Department, Feik School of Pharmacy, University of the Incarnate Word,
San Antonio, TX 78209, USA; banu@student.uiwtx.edu (R.B.); gerding@uiwtx.edu (J.G.);
cefrankl@uiwtx.edu (C.F.)
Department of Chemistry, University of Massachusetts Boston, Boston, MA 02125, USA;
2
william.horton001@umb.edu (W.H.); marianna.torok@umb.edu (M.T.)
Chemistry Department, School of Mathematics, Science and Engineering, University of the Incarnate Word,
3
San Antonio, TX 78209, USA; judavis@uiwtx.edu
Department of Physics and Astronomy and Neuroscience Program, Trinity University, San Antonio,
4
TX 78212, USA; kcheng1@trinity.edu
Anatomy and Physiology Department, School of Osteopathic Medicine, University of the Incarnate Word,
5
San Antonio, TX 78209, USA; nakazwe@uiwtx.edu
Department of Chemistry, Florida A&M University, Tallahassee, FL 32307, USA; mochonab@yahoo.com
6
*
Correspondence: sikazwe@uiwtx.edu
Received: 12 October 2017; Accepted: 13 December 2017; Published: 21 December 2017
Abstract: The objective of this research is to generate leads for developing our ultimate
poly-active molecules with utility in central nervous system (CNS) diseases. Indeed, poly-active
molecules capable of mitigating brain free radical damage while enhancing acetylcholine signaling
(via cholinesterase inhibition) are still being sought for combating Alzheimer’s disease (AD).
We differentiate “poly-active” agents from “multi-target” ones by defining them as single
molecular entities designed to target only specific contributory synergistic pharmacologies in
a disease. For instance, in AD, free radicals either initiate or act in synergy with other
pharmacologies, leading to disease worsening. For this preliminary report, a total of 14
(i.e., 4,5-dimethoxy-2-nitrobenzohydrazide plus 1-(1-benzylpiperidin-4-yl)ethan-1-one) derivatives
were synthesized and screened, in silico and in vitro, for their ability to scavenge free radicals and
inhibit acetylcholinesterase (AChE)/butyrylcholinesterase (BuChE) enzymes. Overall, six derivatives
(
4a
capacity (ORAC) assay. The antioxidant values were either comparable or more potent than the
comparator molecules (ascorbic acid, resveratrol, and trolox). Only three compounds (4d 9a 9c)
, 4d, 4e, 4f, 4g, 9b) exhibited potent (>30%) antioxidant properties in the oxygen radical absorbance
,
,
yielded modest AChE/BuChE inhibitions (>10%). Please note that a SciFinder substance data base
search confirmed that most of the compounds reported herein are new, except 9a and 9c which are
also commercially available.
Keywords: poly-active; antioxidant; hydrazones; isonipecotates; cholinesterase; in silico
1. Introduction
Disease-modifying, anti-Alzheimer’s disease (AD) molecules continue to elude both big and
small pharma discovery approaches. Part of the problem is that AD pathology is underpinned by
Sci. Pharm. 2018, 86, 2; doi:10.3390/scipharm86010002
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errant synergistic or intertwined pharmacologies. Our inclination is that small molecules capable of
modulating disease synergistic or intertwined contributory pharmacologies could more effectively
modify multi-factorial diseases like AD and slow its progression.
Our initial search for potentially poly-active small (Formular Weight (FW) < 500) molecule leads
possessing free radical, acetylcholinesterase (AChE) suppressive activities has thus far produced
compounds with potent radical scavenging capabilities and modest acetylcholine potentiating
properties. The pharmacophores utilized (i.e., 4,5-dimethoxy-2-nitrobenzohydrazide or hydrazones
and 1-(1-benzylpiperidin-4-yl)ethan-1-one or isonipecotates) were pursued due to their reported
multi-pronged attributes, that is, their abilities to donate or accept hydrogen and exert their own
biological effects [1–5]. The fact that they can be readily derivatized with diverse hetero-/non-hetero
aromatic groups to afford new chemical entities possessing advantageous pharmacological profiles is
a bonus [6–9].
The above molecules were designed to structurally contain at least two hetero-aryl functionalities
spaced by a 2 to 3 carbonylated atom linker and electrostatically mimic donepezil (Do, an AChE
inhibitor with clinical utility in all phases of AD) [10–12]. They were synthesized in a parallel approach,
and tested for their radical and AChE inhibitory extents. Antioxidant or radical scavenging capacities
were desired in our molecules because excess reactive oxygen species (ROS), directly (e.g., via protein
and lipid oxidations) and indirectly (e.g., via apoptotic or ß-amyloid mechanisms), ravage neurons in
AD [13–15]. Enzyme inhibition tests were conducted, both in silico (predictively for AChE only) and
in vitro (to confirm for AChE/butyrylcholinesterase (BuChE)). Interestingly, cholinesterases continue
to be drug design targets in this arena, even though their sole role in AD remains somewhat unclear.
For instance, while their inhibition improves the acetylcholine (ACh) signaling for memory/cognition,
the two enzymes also contribute to plaque assembly in AD [12,16].
2. Materials and Methods
2.1. Synthesis
Hydrazone synthesis occurred in two steps (Scheme 1) [
4,5-dimethoxy-2-nitrobenzoate and excess hydrazine monohydrate in absolute ethanol, and afforded
intermediate in yields of 60–70%. In step two, intermediate was condensed with a variety of
aromatic aldehydes 3a to form arylated hydrazones 4a as solids (Figure 1, 70%-quantitative yields).
Exploratory arylated isonipecotates 9a were prepared beginning with the trimethylamine-facilitated
N-benzylation of isonipecotate ethyl ester in toluene (Scheme 2) [ ]. Reaction yields of the benzylated
intermediate were >90%. Ester hydrolysis, under basic reflux, led to the carboxylic acid intermediate,
which was promptly converted to the acyl chloride via the dropwise addition of SOCl2. Finally, acyl
, in step three (Scheme 2), afforded target products
6–8]. Step one involved refluxing methyl
1
2
2
–
k
–k
–
c
5
9
6
7
chloride treatment with appropriate amines 8a
9a–c (60–75% overall yields).
–c
Scheme 1. Reagents and conditions: (i) NH2NH2, EtOH, 60 ◦C, 60–70%; (ii) Hydrazide,
2,3-dihydrobenzofuran-6-carbaldehyde, EtOH, HCl, room temperature, 70%—quantitative.

Sci. Pharm. 2018, 86, 2
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Figure 1. Structures of the synthesized hydrazones (4a–k) and isonipecotates (9a–c). Note that FW
means Formula Weight and MP denotes Melting Point.
Scheme 2. Reactions and conditions: (i) Benzyl chloride, Et3N, toluene, room temperature, > 90%; (iia)
NaOH, H2O, CH3OH, reflux, not isolated; (iib) CH2Cl2, SOCl2, reflux, not isolated; (iiia) Et3N, NH2-Ar,
dioxane or CH2Cl2, reflux, 60–75%; (iiib) HCl-ether, >80%.
2.2. Oxygen Radical Absorbance Capacity (ORAC) Assay
We needed to screen our compounds for their direct radical scavenging capabilities in both lipid
and aqueous environments. To that end, we utilized the oxygen radical absorbance capacity (ORAC
)
assay, which measures peroxyl radical scavenging via hydrogen atom transfer (HAT) or electron
transfer (ET) to the existing or pre-formed radical. Assays details are well reported [17
utilized known conditions for our assay and 2,2 -azobis(2-amidino-propane) dihydrochloride (AAPH)
,
18]. Briefly, we
0
as the oxidant or peroxyl radical ROS generator [19
solution was prepared in 75 mM phosphate buffer (pH = 7.4, kept at 4 C), diluted with the same
buffer to a concentration of 0.0816 µ
M, and incubated at 37 ◦C for 15 min before assaying. A fresh
–
21]. In our hands,◦4.19
µM fluorescein stock
153 mM AAPH solution in the said buffer was prepared, kept on ice, and used for 4 h at the most.
Then, 10 mM solutions of trolox in ethanol, 10 mM solutions of ascorbic acid in water, and 50 mM stock
solutions of test compounds in DMSO were prepared, and each was diluted to 80
Subsequently, 25 L of each diluted stock solution or 25 L of ethanol with 0.16% DMSO in case of
control (no test or reference compound) was plated with 150 L of fluorescein solution, and 25
of the above AAPH solution was added to all wells except those for maximum fluorescence control.
Thus, the test compounds (4a 9a , and donepezil or Do) and the reference compounds (ascorbic
µM with ethanol.
µ
µ
µ
µL
–k
,
–c
acid, resveratrol, and trolox) were all tested at final concentrations of 10 µM in the assay.
The maximal fluorescence intensity was obtained by a SpectraMax i3x microplate reader equipped
with SoftMax Pro 6.5.1 (Molecular Devices, Sunnyvale, CA, USA) software at an emission wavelength
of 520 nm with a preset excitation wavelength of 485 nm. Measurements were taken kinetically
◦
every 2 min for 60 min at a constant temperature of 37 C. Plates were shaken for 5 s before each

Sci. Pharm. 2018, 86, 2
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reading. Measurements were run on multiple plates in triplicate sets. Plates were sealed with
a transparent cover to prevent evaporation. Background of the AAPH solution with appropriate
amount of DMSO, ethanol, and buffer, but no fluorescein was taken in every plate and used as a blank
for all the wells tested. Percent radical scavenging activity was calculated using the expression:
[(AUCt − AUCc)/AUCf_max] × 100%, where AUCt is the net area under the fluorescence curve
obtained in the presence of the test/reference compounds, AUCc is the net area under the fluorescence
curve obtained for the control sample that contained no antioxidant (no test/reference compound), and
AUCf_max is the net area under the fluorescence curve obtained for the maximum fluorescence control
sample that contained no radical and thus had the maximum amount of fluorescein dye. The net area
(AUC) under the fluorescence curves was determined using the following equation:
ꢀ
ꢁ
fi
f0
f30
f0
Net AUC = 0.5 +
+
0.5 ∗
∑
0−29
where f0 is the measured fluorescence intensity at time 0 and fi is the measured fluorescence intensity
at time i. The ORAC assay percent radical scavenging activities are reported in Figure 2.
100
90
ORAC
80
70
60
50
40
30
20
10
0
Figure 2. The percentage (%) of radical scavenging of three reference antioxidants (ascorbic acid, resveratrol,
and trolox), hydrazones (4a–k), isonipecotates (9a–c), and donepezil (Do) determined in the oxygen radical
absorbance capacity (ORAC) assay after 60 min. Data are expressed as means of the % radical scavenging
± standard deviation (SD), where the number of independent repeats is n = 3 [22].
2.3. In Silico AChE Inhibition
Since the said compounds were designed to mimic Do’s structural/electronic and therefore
pharmacologic behaviors, molecular dockings of all fourteen ligands were conducted against AChE
only. The crystal structure of AChE was derived from the Protein Data Bank database (PDB ID:
1EVE) [11]. Before docking, water molecules and the embedded Do ligand were removed from the
AChE protein structure. Non-polar hydrogens were added to the protein using AutoDock Tools
(Version 1.5.6) software and the correct protonation state of each ligand was determined at pH 7.4
using MarvinSketch (Version 17.2.27 ChemAxon, Cambridge, MA, USA) [23]. To create the optimized
3D structures, we used obconformer—a molecular mechanics modeling program based on the force
field MMFF94 from Open Babel [24]. Docking of the ligands to AChE was performed using AutoDock
Vina (Version 1.1.2) [25]. Flexible ligand conformations were used in all dockings. For search space,
a rectangular box of size 28.5
embedded Do was used.
×
18.75
×
18.75 Å3 with its geometrical center set to that of the originally
Nine different conformations (1 to 9) with the binding energies sorted from the lowest to highest
binding energy were obtained from the molecular docking, and the energies of the representative
ligands, Do, 9a, and 9b, are shown in Figure 3. Also, the average and the standard error of each ligand
are demonstrated for the representative ligands. The average and the minimum binding energies
of all fourteen compounds were evaluated, and their values are represented as the binding energy
differences, i.e., the binding energy of each ligand minus the binding energy of Do; for both, average

Sci. Pharm. 2018, 86, 2
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and minimum energies are also indicated in Figure 3. It is clear that 9a exhibited the lowest difference
in binding energy versus other compounds. Figure 4A–C overlaid structures show the predicted
conformations of representative ligands Do, 9a, and 9b, respectively. Essentially, nine structures of each
ligand are superimposed and the ones with the lowest binding energies are highlighted in black, pink,
and green, accordingly. Figures 4D and 4E, respectively illustrate the lowest energy structures of 9a and
9b versus Do in AChE active site/gorge. Notably, the lowest energy structure of our docked Do closely
matched that of the reported crystal structure of embedded Do
proximities of the following protein residues with various groups of Do: (1) Trp279 to the indanone
ring of Do via interactions at the proposed entrance to the gorge of AChE; (2) PhE330 and Tyr121
to the nitrogen of the piperidine ring of Do via cation- and hydrogen bonding, respectively, in the
stacking at the bottom of the gorge,
[11]. Specifically, we observed close
π-π
π
middle of the gorge; and (3) Trp84 to the benzyl ring via
as proposed previously [11].
π-π
4.0
Conformational State
7
Ave
1
2
3
4
5
6
8
9
-9
-10
-11
-12
3.0
2.0
1.0
0.0
-1.0
Do 9a 9b
average
minimum
Do 4a 4b 4c 4d 4e
4f 4g 4h
4i
4j
4k 9a 9b 9c
Figure 3. Summary of binding energies obtained for all compounds reported herein including
comparative conformations of Do versus compounds 9a and 9b.
Figure 4. Computational binding of compounds at acetylcholinesterase (AChE). (
A
–C
) represent the
nine predicted conformations while (D,E) refer to the lowest energy binding orientations obtained
by docking.

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2.4. In Vitro AChE/BuChE Inhibition
To determine compound selectivity, both AChE and BuChE inhibitory studies were undertaken.
The two assays were conducted using modified Ellmann’s procedures [26 29]. Electric eel AChE
–
(catalog number: C2888-500UN) and equine serum BuChE (catalog number: C4290-1KU) were
purchased from (Sigma-Aldrich, St. Louis, MO, USA). Enzyme aliquots of 6 U/mL were prepared in
◦
20 mM HEPES buffer (Ph = 8.0) containing 0.1% TritonX-100, stored at
−
20 C until use, when
they were thawed and diluted 20X with 100 mM phosphate buffer (pH = 8.0). Subsequently,
10 mM Stock solutions of inhibitors (test compounds, galantamine (Ga) and Do) were prepared
in DMSO and then diluted to 0.15 mM through a co-solvent method by adding 145.5
µL of 0.1 M
phosphate buffer (pH = 8.0) and 150
µ
L of acetonitrile to 4.5
µ
L of 10 mM inhibitor stock solution.
0
5,5 -dithio-bis-(2-nitrobenzoic acid) or DTNB, also called Ellman’s reagent, stock solution of 0.4341 mM
in 100 mM phosphate (pH = 8.0 buffer) was also prepared. Finally, depending on the assay,
acetylthiocholine or butyrylthiocholine stock solutions (4.124 mM in 100 mM phosphate buffer,
pH = 8.0) were made. These stock solutions were used in the ensuing enzymatic reactions in 96 wells.
Ultimately, each well comprised a final assay volume of 150
DTNB, 0.02 unit/mL AChE or BuChE, 0.55 mM acetylthiocholine or butyrylthiocholine, and 2
inhibitor (except in the case of the control, which had no inhibitor) for AChE or 10 M inhibitor (except
µL and the following ingredients: 0.34 mM
µM
µ
in the case of the control, which contained no inhibitor) for BuChE. Assays were also carried out
with a blank solution containing all components except the enzyme and inhibitor so as to account for
non-enzymatic reactions.
Measurements were run on multiple plates, in triplicates. The substrates acetylthiocholine and
butyrylthiocholine were cleaved by their respective enzymes, generating thiol groups detected via their
reaction with the colorimetric Ellman’s reagent, DTNB. Initial rate measurements were performed at
◦
37 C using a VersaMax microplate reader with SoftMax Pro 5 software (Molecular Devices, Sunnyvale,
CA, USA) and collecting absorbances at 412 nm every 15 s for 15 min. Percent inhibitions of the enzyme
activity due to the presence of test compounds with respect to the control were calculated by the
following expression: [(v0 − vi)/v0] × 100, where
vi and v0 are the rates calculated in the presence and
absence of an inhibitor. AChE and BuChE act independently to hydrolyze/deactivate acetylcholine,
and their inhibition leads to enhancements in the levels and activity of ACh. Enzyme inhibition data
obtained from test and reference molecules are displayed in Figure 5.
100
AChE
90
80
70
60
50
40
30
20
10
0
BuChE
-10
Ga 4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 9a 9b 9c Do
Figure 5. Respective enzyme inhibitions upon exposure to test and reference compounds (galantamine
or Ga and Do). All compounds were tested at 2 M (AChE) using 0.02 U/mL enzyme quantities.
µ
Data are expressed as means of the % AChE inhibition ± SD, n = 3. BuChE: butyrylcholinesterase.
3. Results and Discussion
Overall, 11 4,5-dimethoxy-2-nitrobenzohydrazide or hydrazone plus three 1-(1-benzylpiperidin-
4-yl)ethan-1-one or isonipecotate derivatized small molecules (Figure 1, FWs <500) were synthesized
and preliminarily evaluated at 10
inhibition, using established techniques. Compounds 4a
(>30%) in the ORAC assay; that is, they performed at or better than ascorbic acid. In fact, 4a
µ
M for free radical scavenging abilities, and at 2
4d , and 9b potently scavenged radicals
4d, and
µM for cholinesterase
,
–g
,

Sci. Pharm. 2018, 86, 2
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4g performed comparable to reference compounds trolox and resveratrol. The ORAC differentiation in
antioxidant capability is significant because this assay employs radicals with practical relevance in
living organisms.
In terms of cholinergic activity, most analogs poorly inhibited (<10%) AChE and BuChE.
The exception was isonipecotates 9a and 9c, which modestly inhibited (10–20%) both enzymes.
As expected, the reference compounds (Ga and Do) differentially inhibited AChE (almost 60% for Ga
,
and 98% for Do) and BuChE (by about 30% by Ga, and 60% for Do). Despite the low enzyme inhibitory
activities, we were encouraged by a finding that a linear correlation existed when calculated binding
free energy (kcal/mol) differences and experimentally derived % AChE binding inhibition differences
were plotted, as illustrated in Figure 6. This correlation was meaningful because it confirmed that
our predictive computational model for AChE binding was on the right path and implied that the
designed compounds yielded useful leads whose cholinergic shortfall could be improved by SAR
(structure activity studies). We now know that
for AChE/BuChE inhibition. Rather, a combination of
may prove useful.
π
-
π
stacking may not be the only essential SAR element
interactions plus H-bonding or polar groups
π-π
Figure 6. Free Energy vs. % AChE binding inhibition differences.
4. Conclusions
Taken together, this preliminary report indicates that we have generated six (4a
,
4d, 4e, 4f, 4g, 9b)
good leads with strong antioxidant and minimal AChE inhibition activities. SAR studies and additional
pharmacological evaluations will be undertaken to determine if these molecules meet our ultimate
poly-active molecules design goal. Note that the essence of our approach to drug design is to develop
molecules that can modulate synergistic disease pharmacologies—ROS reductions are a good starting
point. Regarding any additional experimental details/data (NMR, Mass, etc.), this manuscript is simply
a short communication or preliminary report on compounds whose synthesis is already well established
and appropriately documented in the included references.
Author Contributions: Banu Rukhsar, Jason Gerding, Cynthia Franklin, and Muziya Nakazwe were involved in
the synthesis/cytotoxicity and other in vitro characterizations of the reported compounds; Donald Sikazwe
designed the compounds and wrote the manuscript; William Horton and Marianna Török conducted the
antioxidant and cholinesterase experiments; Julian Davis synthesized compounds 9a–c; Kwan H. Cheng carried
out the in silico cholinesterase modeling while Bereket Mochona advised and performed additional in vitro tests
on the compounds. All authors provided input on the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.

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