Multi-targeted dihydrazones as potent biotherapeutics

Article abstract of DOI:10.1016/j.bioorg.2018.08.024

Hydrazone compounds were considered as a useful moiety in drug design development. Therefore, these studies were aimed at the synthesis of new dihydrazones and were screened for their in vitro H⁺/K⁺-ATPase and anti-inflammatory activities. The results revealed that compounds 9 (22 ± 0.62 μg/mL), 10 (26 ± 0.91 μg/mL), 15 (24 ± 0.44 μg/mL), 16 (28 ± 0.63 μg/mL), 17 (12 ± 0.38 μg/mL), 18 (14 ± 0.47 μg/mL), 19 (26 ± 0.54 μg/mL), 20 (16 ± 0.41 μg/mL), 25 (06 ± 0.68 μg/mL) and 26 (08 ± 0.43 μg/mL) showed excellent H⁺/K⁺-ATPase activity and their IC₅₀ value were lower than the standard drug Omerazole (48 ± 0.12 μg/mL). Compounds 5 (28 ± 0.65 μg/mL), 6 (24 ± 0.61 μg/mL), 7 (28 ± 0.64 μg/mL), 8 (26 ± 0.45 μg/mL), 11 (30 ± 0.74 μg/mL), 12 (28 ± 0.40 μg/mL), 13 (32 ± 0.24 μg/mL), 14 (30 ± 0.55 μg/mL) and 21 (08 ± 0.47 μg/mL), 22 (12 ± 0.47 μg/mL), 23 (10 ± 0.51 μg/mL) and 24 (14 ± 0.84 μg/mL) showed better anti-inflammatory activity compared to standard indomethacin (44 ± 0.15 μg/mL). The structure activity relationship (SAR) showed that, electron donating groups (OH, OCH₃) favored the H⁺/K⁺-ATPase and antioxidants activity, whereas, electron withdrawing groups (F, Cl, Br and NO₂) favored the anti-inflammatory activity. Furthermore, molecular docking study was performed to investigate the binding interactions of the most active analogs with the active site of H⁺/K⁺-ATPase enzyme. Compounds 25 (G-score = ?9.063) and 26 (G-score = ?8.977) showed the highest docking G-scores for H⁺/K⁺-ATPase inhibition activity.

Full text of DOI:10.1016/j.bioorg.2018.08.024

Accepted Manuscript
Multi-targeted dihydrazones as potent biotherapeutics
Chen Li, M.B. Sridhara, K.P. Rakesh, H.K. Vivek, H.M. Manukumar, C.S.
Shantharam, Hua-Li Qin
PII:
S0045-2068(18)30742-9
DOI:
https://doi.org/10.1016/j.bioorg.2018.08.024
YBIOO 2481
Reference:
Bioorganic Chemistry
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Accepted Date:
19 July 2018
15 August 2018
20 August 2018
Please cite this article as: C. Li, M.B. Sridhara, K.P. Rakesh, H.K. Vivek, H.M. Manukumar, C.S. Shantharam, H-
L. Qin, Multi-targeted dihydrazones as potent biotherapeutics, Bioorganic Chemistry (2018), doi: https://doi.org/
10.1016/j.bioorg.2018.08.024
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Multi-targeted dihydrazones as potent biotherapeutics
Chen Li1, M. B. Sridhara2*, K. P. Rakesh 1*, H. K. Vivek3, H. M.
Manukumar4, C. S. Shantharam5 and Hua-Li Qin1*
1Department of Pharmaceutical Engineering, School of Chemistry, Chemical Engineering
and Life Science, Wuhan University of Technology, 205 Luoshi Road, Wuhan, 430070, PR
China
2Department of Chemistry, Rani Channamma University, Vidyasangama, Belagavi-591156,
Karnataka, India
3Analytical Research and Development, Syngene International Ltd, Biocon Park,
Bommasandra Industrial Estate, Bangaluru-560099, Karnataka, India
4Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysuru-
570006, Karnataka, India
5Department of Chemistry, Pooja Bhagavath Memorial Mahajana Education Centre, Mysuru-
570016, Karnataka, India
Abstract
Hydrazone compounds were considered as a useful moiety in drug design
development. Therefore, these studies were aimed at the synthesis of new dihydrazones and
were screened for their in vitro H+/K+-ATPase and anti-inflammatory activities. The results
revealed that compounds 9 (22±0.62 µg/mL), 10 (26±0.91 µg/mL), 15 (24±0.44 µg/mL), 16
(28±0.63 µg/mL), 17 (12±0.38 µg/mL), 18 (14±0.47 µg/mL), 19 (26±0.54 µg/mL), 20
(16±0.41 µg/mL), 25 (06±0.68 µg/mL) and 26 (08±0.43 µg/mL) showed excellent H+/K+-
ATPase activity and their IC50 value were lower than the standard drug Omerazole (48±0.12
µg/mL). Compounds 5 (28±0.65 µg/mL), 6 (24±0.61 µg/mL), 7 (28±0.64 µg/mL), 8
(26±0.45 µg/mL), 11 (30±0.74 µg/mL), 12 (28±0.40 µg/mL), 13 (32±0.24 µg/mL), 14
(30±0.55 µg/mL) and 21 (08±0.47 µg/mL), 22 (12±0.47 µg/mL), 23 (10±0.51 µg/mL) and 24
(14±0.84 µg/mL) showed better anti-inflammatory activity compared to standard
indomethacin (44±0.15 µg/mL). The structure activity relationship (SAR) showed that,
electron donating groups (OH, OCH3) favored the H+/K+-ATPase and antioxidants activity,
whereas, electron withdrawing groups (F, Cl, Br and NO2) favored the anti-inflammatory
1

activity. Furthermore, molecular docking study was performed to investigate the binding
interactions of the most active analogs with the active site of H+/K+-ATPase enzyme.
Compounds 25 (G-score = -9.063) and 26 (G-score = -8.977) showed the highest docking G-
scores for H+/K+-ATPase inhibition activity.
Key words: Dihydrazones; H+/K+-ATPase; anti-inflammatory activity; antioxidants; docking
study.
*Corresponding authors
E-mail: rakeshasg@gmail.com
Tel: +91 9964100602
E-mail: sridhara.mb@gmail.com
Tel: +91 9663983459
Email: qinhuali@whut.edu.cn
Fax: +86 27 87749300.
1. Introduction
The continuous active research in the area of chemistry and biology helps
pharmaceuticals to reduce the pain and other associated impairments by medicine, which is
reported 90% as statistics of world health organization (WHO). The advanced knowledge of
endogenous nociceptive and antinociceptive systems, different pain associated diseases such
as rheumatoid arthritis and advanced cancers are away from targeted or particular medicine to
cure till date [1]. Currently available nonsteroidal anti-inflammatory drugs (NSAIDs) like
ibuprofen, indomethacin and naproxen exhibit gastric toxicity. Long-term use of these drugs
has been associated with gastrointestinal (GI) ulceration, bleeding and nephrotoxicity [2].
The pharmacological activity of NSAIDs is related to the suppression of prostaglandin
biosynthesis from arachidonic acid by inhibiting cyclooxygenases (COXs) [3,4]. The
utilization of NSAIDs drugs without the consequence of GI toxicity can increase the adverse
effect in the body. With this knowledge, the safety measures should be taken into account
during the chemical approaches being involved in synthesizing NSAIDs.
2

Gastric and duodenal ulcers are common diseases of the gastrointestinal tract with
high clinical incidence rates, which may result in severe superior gastrointestinal bleeding.
They may be caused by an imbalance between aggressive and defensive forces in the stomach
and duodenum. The reduction of acid secretion has been proven to be a useful means for
promoting the healing of ulcers [5,6]. As a result, H+/K+ ATPase could be a therapeutic target
for various acid-related diseases. Although a number of H+/K+ ATPase inhibitors are
available on the market, there is always a need for new agents with even better efficacy and
safety profiles.
Hydrazones containing azomethine (-NH-N=CH) protons constitute a vital class of
compounds for new drug development [7]. Several heterocyclic hydrazones were reported to
possess various biological activities viz. antimicrobial [8], anti-inflammatory [9], antioxidants
[10], analgesic [11], anti-candida [12], antitubercular [13], anticancer [14], antiproliferative
[15], and α-glucosidase [16], activities. Particularly, hydrazone moiety attached to
heterocyclic systems was shown to offer enhanced activity [17,18]. Based on this background
we planned to design and synthesis of new dihydrazone derivatives affecting aromatic
hydrazones as an important pharmacophore to development of new future drugs for anti-
inflammatory as well as H+/K+ -ATPase agents (Figure 1).
Based on the above interpretation and in continuation of our drug development
program [19-23], herein we report the synthesis of dihydrazones and were screened for their
in vitro H+/K+ -ATPase, antioxidant and anti-inflammatory activities. Moreover, we carried
out in silico molecular docking approaches to validate the H+/K+-ATPase inhibitor agents of
synthesized compounds.
2. Results and Discussion:
2.1 Chemistry:
Syntheses of the dihydrazones were achieved according to the procedures illustrated
in Scheme 1. Isophthalic acid (1) was methylated using trimethylsilylchloride (TMS-Cl) and
3

methanol at room temperature to get isophthalic acid dimethyl ester (2) which upon reaction
with an excess of hydrazine hydrate afforded the corresponding isophthalic hydrazides (3).
The hydrazones (4-36) were obtained by reacting 3 with different aromatic or aliphatic
aldehydes in presence of catalytic amount of glacial acetic acid. All the derivatives were
obtained in excellent yield and the methods employed are very simple. The structures of all
1
the newly synthesized compounds including intermediates were confirmed by HNMR,
13CNMR and mass spectral analysis (Supplementary material). The formation of methyl
esters (2) was confirmed by the appearance of a singlet at 3.83δ for -OCH3 and absence of
1
1
COOH proton peak at 12.10δ in the HNMR spectrum. In the H NMR spectrum of 3, two
singlet signals displayed at 9.79 ppm and 4.50 ppm corresponding to the NH and NH2
protons, respectively. Moreover, the absence of one signal at 52.3 ppm of –OCH3 in 13C
NMR spectrum confirms the formation of hydrazide 3 from methylester 2. The final
1
13
compounds 4-36 were confirmed by their H NMR, C NMR and mass spectrum analysis.
1H NMR spectrum of 4 showed a singlet for –NH proton at 12.03 ppm and disappearance of
the peak for NH2 proton confirms its formation. Further, it showed a singlet at 8.48 ppm for
the newly formed azomethine proton (-N=CH-) confirms its formation of hydrazones. All
final compounds 4-36 showed carbonyl (C=O) signal in the region of 162–164 ppm and
13
azomethine carbon (-N=CH-) signal in the region of 146–148 ppm in their C NMR spectra
clearly confirms their formation hydrazones. Further, compound 4 showed a M+1 peak at
371.2414 in their HRMS confirms the formation of final products.
2.2 Biology:
2.2.1 H+/K+-ATPase activity:
In this study, we aimed to investigate the pharmacological potential of synthesized
compounds were synthesized and considering the medicinal use of synthetic chemistry. To
find out potential H+/K+-ATPase inhibition candidates, all the newly synthesized compounds
4-36 were screened for their in vitro H+/K+-ATPase inhibition activity and their results were
4

tabulated in Table 1 and results were compared to standard Omeprazole. Among the
derivatives, compound 4 with no substitution on the phenyl ring showed activity with IC50 =
88 μg/mL, which is less potent than omeprazole IC50 = 48 μg/mL. It can be inferred that
compounds without substitution on the phenyl ring showed less activity. Therefore, the effect
of substituent on the phenyl ring was further investigated. Dihydrazones with different
electron donating (OH and OCH3), electron withdrawing (Cl, NO2, F and Br), groups on the
phenyl ring and replacing phenyl ring with some aliphatic and heterocyclic substituents were
also evaluated. It is found that, when hydroxy and/or methoxy groups substituted on the
phenyl ring, the corresponding derivatives showed potent H+/K+-ATPase inhibition activity
[24]. According to the old hypothesis, acid secretion was thought to be the sole cause of ulcer
formation and reduction in acid secretion was thought to be the major approach towards
therapy. However, in the light of recent evidences this concept has changed. With the
knowledge of ulcer research, nowadays during targets have been identified and compounds
are synthesized to decrease the acid secretion system as potential targets of this condition. In
our previous H+/K+-ATPase inhibition studies of quinazolinones and benzo[d]thiazole
derived hydrazones showed potent H+/K+-ATPase inhibition activity [6, 20]. But in this paper
we thinking on the different way and designed the molecules without any biologically active
heterocycles. On this basis we design and synthesis of simple dihydrazones and they screened
for in vitro antiulcer activity and showed excellent inhibitory activity compared to our
previously published quinazolinone and benzo[d]thiazole hydrazones [6, 20]. Compounds 9,
10, 15, 16, 17, 18, 19, 20, 25 and 26 showed excellent H+/K+-ATPase inhibition activity with
IC50 values of 22, 26, 24, 28, 12, 14, 26, 16, 06 and 08 µg/mL respectively, which were much
better than the standard Omeprazole (IC50 = 48 µg/mL). The inhibition activity increased
along with increased number of hydroxyl and methoxy groups. With six hydroxyl (25) and
six methoxy (26) groups on the phenyl ring, the derivatives showed potent H+/K+-ATPase
inhibition activity with IC50 values of 06 and 08 µg/mL respectively and the activity trends
5

were 2 OH or OCH3 ˃ 4 OH or OCH3 ˃ 6 OH or OCH3. Further, we were also interested in
designing the molecule, by replacing the aromatic and aliphatic aldehyde (27-29) and with
some heterocyclic aldehydes (31-33) and test for their H+/K+-ATPase inhibition activity.
Furan (31), thiophene (32) and pyrazole (33) derived analogs showed moderate H+/K+-
ATPase activity. Derivatives with electron withdrawing groups (Cl, NO2, F and Br) on
phenyl ring and aliphatic analogs (27-29) showed least antiulcer activity.
2.2.2 Anti-inflammatory activity:
Inflammation is a response of immune system that activates many enzymatic and
cellular processes to protect the body from all kinds of trauma. To address this, the
synthesized all compounds were evaluated for their anti-inflammatory activity using in vitro
human erythrocytes model [25]. A significant number of compounds have been identified and
showed excellent to moderate inhibitory activity compared to standard drug indomethacin.
IC50 was determined for the compounds showing more than 50% inhibition concentration
(Table 1). The compounds 5, 6, 7, 8, 11, 12, 13, 14, 21, 22, 23 and 24 showed excellent
activity with IC50 values of 28, 24, 28, 26, 30, 28, 32, 30, 08, 12, 10 and 14 µg/mL
respectively, much better than the standards indomethacin (IC50 = 44 µg/mL). The electron
withdrawing groups were most favorable to show anti-inflammatory activities [26] exhibited
by two or more electron withdrawing groups (21-24) present in benzene ring which are
involved in highly potent anti-inflammatory activity compared to the single electron
withdrawing groups (5-8 and 11-14). Electron withdrawing (Cl, NO2, F and Br) group
presents in para position (5-8) showed more anti-inflammatory activity compared to ortho
position (11-14). Electron donating groups (OH and OCH3) shows the least anti-
inflammatory activity.
2.2.3 Inhibition of COX-2 enzyme activity
Modulation of the activity of pro-inflammatory enzymes is one of the most important
mechanisms of action for synthetic compounds. Pro-inflammatory enzymes, such as cytosolic
6

phospholipase A2 (cPLA2), cyclooxygenases (COX), lipoxygenases (LOX), and NO
synthase (NOS), produce very potent inflammatory mediators, and therefore their inhibition
contributes to the overall antiphlogistic potential of synthetic derivatives in the drug
discovery programs compared to the natural compounds [27, 28]. Compounds 1-36 were
studied for inhibition of COX-2 [29] at a concentration of 30 μM in order to compare the
effects of each of the compounds and to select the most promising active substance, using
ibuprofen as a positive control. The compounds 5, 6, 7, 8, 11, 12, 13, 14, 21, 22, 23 and 24
showed significant effects on COX-2 inhibition, comparable with the reference compound.
Therefore, further analyses of selected promising 5, 6, 7, 8, 11, 12, 13, 14, 21, 22, 23 and 24
compounds were carried out to determine the IC50 values against both COX-2 to assess the
promising effect of selectivity of each compound (Table 1). Among them, the compounds 21
(06±0.10), 22 (10±0.75), 23 (08±0.51) and 24 (06±0.73) showed potent COX-2 inhibitors.
This indicates that, these inhibitors involved in the decrease in the sign of inflammation
accordance with literature reports [30, 31].
2.2.4 Antioxidant activity
In vitro antioxidant activities profile of the synthesized dihydrazones (4-36) were
evaluated by (i) 1,1-diphenyl-2-picryl-hydrazyl (DPPH) assay which is a rapid and
convenient technique for screening the antioxidant activities of the antioxidants, (ii) 2,2-
azinobis-3-ethylbenzothiazoline-6-sufonic acid (ABTS) cation radical assay which is a
conventional and excellent model for assessing the antioxidant activities of hydrogen
donating and chain breaking antioxidants [32] and (iii) N,N-dimethyl-p-phenylenediamine
dihydrochloride (DMPD) cation radical assay which is similar to the DPPH radical
scavenging assay. The values of IC50, the effective concentration at which 50% of the radicals
were scavenged, were calculated to evaluate the antioxidant activities. A lower IC50 value
indicated greater antioxidant activity. IC50 values of lower than 10 mg/mL usually implied
7

effective activities in antioxidant properties [33]. The IC50 of ascorbic acid (AA) and gallic
acid (GA) was also determined for comparison. The results were revealed in Table 1.
Most of the synthesized compounds showed potent antioxidant activities. Compounds
9, 10, 15, 16, 17, 18, 20, 25 and 26 showed excellent radical scavenging activities with IC50
values 28, 34, 30, 36, 20, 22, 20, 08 and 12 µg/mL respectively in DPPH assay much better
than the standards ascorbic acid (IC50 = 44 µg/mL) and gallic acid (IC50 = 48 µg/mL). In
ABTS+ radical scavenging assay, the compounds 9, 10, 15, 16, 17, 18, 20, 25 and 26 showed
potent antioxidant activity with IC50 values 26, 36, 32, 38, 16, 24, 16, 10 and 14 µg/mL
respectively which is much better than the commercial standards ascorbic acid (IC50 = 48
µg/mL) and gallic acid (IC50 = 42 µg/mL). The compounds 9, 10, 15, 16, 17, 18, 20, 25 and
26 also exhibited striking antioxidant activity with IC50 values 30, 34, 32, 36, 20, 18, 18, 12
and 10 µg/mL respectively which is better than the standards ascorbic acid (IC50= 42 µg/mL)
and gallic acid (IC50 = 46 µg/mL) in DMPD assay.
To study the structure activity relationship (SAR), compounds having –OH (phenolic)
and –OCH3 (anisole) groups in the phenyl ring (9, 10, 15, 16, 17, 18, 20, 25 and 26) were
found to be the most potent antioxidants [34]. The number of electron donating groups (OH
and OCH3) increases on the phenyl ring, the activity also increases (25 and 26). The
compounds 25 and 26 showed most potent antioxidants compared to the other electron
donating compounds. Due to the presence of three hydroxy or three methoxy groups presents
on the both side of phenyl rings. Compound 32 showed good antioxidant activity in all the
tested antioxidants methods due to the presence of sulpur atom in the molecule. The
compounds with electron withdrawing Cl, F, NO2 and Br substituents on the phenyl groups
(5-8, 11-15 and 21-24) showed least antioxidants activity [34]. Compounds containing
aliphatic analogs (27-29) showed nil antioxidants activity in all the three tested antioxidants
methods.
2.2.5 Docking studies:
8

In order to determine the possible binding modes for compound 25, molecular
docking studies were performed for ligands 2-36. The structure of the protein from Daboia
russelii (PDB ID: 1E9Y) was employed for docking studies. Analysis of the interaction mode
for the lower energy docking pose among the ensemble indicated that compound 25
interacted with Ala365, Gly279, Asp223 and His221 by π-π stacking (Fig. 2). Importantly,
binding of ligand 25 near the α-helical random coil clearly suggests that it had interaction
with His221, which resides near the random coil [35].
Likewise, to rationalize the inhibition seen for Na+/K+ ATPase and o gain insights
into the possible binding modes, rigid-body docking studies of the compounds was carried
out with the receptor structure. Crystal structure of Na+/K+-ATPase from Squalus acanthias
(Spiny Dogfish) (PDB ID: 2ZXE) was used for molecular docking studies as a prototype
instead of the structure of the protein from human for ease of target structure preparation.
Molecular docking studies showed an interacting map of Na+/K+-ATPase enzyme with
compound 25 (Fig 3). Based on XP glide score, compounds 25 and 26 showed a promising
scoring function, when compared to other structurally related compounds (See supporting
information Table 2). Additionally, the low E model value indicated that the binding affinity
between protein and ligand is energetically favourable [36]. Pharmacodynamics parameters
play an important role in determining the success of a lead candidate for further therapeutic
development.
3. Conclusion:
In conclusion, in an attempt of developing a new class of anti-inflammatory,
antioxidants and H+/K+-ATPase agents, novel isophthalic dihydrazone analogs were
efficiently synthesized in good yield. Bioassay results revealed that compounds 9, 10, 15-20,
25 and 26 with OH and OCH3 groups on phenyl ring (electron donating) showed excellent
H+/K+-ATPase and antioxidants activities than the corresponding standard drugs. Compounds
5-8, 11-14 and 21-25 with Cl, NO2, F and Br on phenyl ring (electron withdrawing) also
9

showed excellent anti-inflammatory activity. Furthermore, molecular docking studies were
performed for all the synthesized compounds, among which compounds 25 and 26 showed
the highest docking G-scores for H+/K+-ATPase inhibition activity.
4. Biological activity
4.1 Gastric H+/K+-ATPase activity
Isolation of parietal cells from sheep stomach
The fundic stomach portion of sheep was collected soon after sacrificing and was rinsed with
Krebs ringer buffer (250 mM sucrose, 2 mM MgCl2, 1 mM EGTA and 2 mM Hepes-Tris of
pH 7.4). The upper layer was pinned with the help of needles on the dissection table. Mucosal
scrapings were hanged in 10 volumes of Krebs ringer buffer (pH = 7.4) containing sucrose
(250 Mm) and homogenized with 20 strokes of a mortar driven Teflon pestle homogenizer.
The tissues were discarded and the filterate was subjected to sub cellular fractionation. The
pellets obtained were dissolved in 2 mL of sucrose-EGTA buffer and used as enzyme sample.
Protein estimation
Protein was calculated using the Lowry method [37] and bovine serum albumin as
standard (0-75 µg). Eight clean and dry test tubes were taken and aliquots of various
concentrations of the synthesized derivatives were made. To the 7th and 8th test tube, the
unknown sample (5 µL and 10 µL of the cells isolated from the sheep’s stomach) for which
the protein content was added. In every test tube, the solution was made up to 1 mL by the
addition of distilled water, followed by the addition of 5 mL of Lowry’s reagent. All the test
tubes are incubated at rt for 8-10 min and 0.5 mL of Folin-Ciocalteu reagent was added and
again incubation at rt for 30 min. Absorbance of each solution was recorded at 670 nm
against the blank solution. A graph was plotted by using concentration of protein on X-axis
and OD on Y-axis. From the standard graph obtained, the unknown concentration of protein
sample was calculated and found to contain 21 mg of protein per 8 g of tissue homogenate.
Inorganic phosphorus estimation
10

Inorganic phosphorus was estimated according to Fiske Subbarow’s method [38].
Aliquots of working standard solution (40µg/mL) were added into 8 fresh and dry test tubes
in the volume of 0 to 1mL; 5 µL and 10 µL of the enzyme sample were taken in test tube 7th
and 8th respectively. The volumes of all test tubes were made up to 8.6 mL using 10% TCA.
Ammonium molybdate (1mL) and ANSA reagent (0.4 mL) were added to all test tubes. All
test tubes were allowed to stand for 10 min at rt. Then the color was developed and the λmax at
660 nm were recorded.
4.2 ATPase activity
ATPase activity was determined using reported method [39, 40]. Basal Mg2+ dependent
ATPase activity was calculated in 1 mL reaction mixture comprising of 2 mmol/L ATP and
-
50 mmol/L Tris-HCl buffer (pH=7.5). K+ stimulated and HCO3 stimulated ATPase activity
were tested in the basal medium. The ATPase reaction was started by the addition of the
substrate (ATP), carried out at 37 °C for 10-15 min and the terminated by the addition of 1
mL cold 20% TCA. Liberated inorganic phosphate from ATP was estimated using literature
method [40].
Activity of the enzyme sample in the presence of ATP is 0.066 µmoles.
Activity of the enzyme sample in the absence of ATP is 0.042 µmoles.
100% activity of the enzyme is 0.066 µmoles-0.042 µmoles = 0.024 µmoles.
4.3 Anti-inflammatory activity:
Human erythrocyte suspension
The human blood was purchased from public hospital, Mysore, India and collected in
heparinzed vacutainer. The collected healthy human blood was washed with 0.9% saline and
centrifuged for 10 minutes at 3000 rpm. The packed cells were washed with 0.9% saline and
40% v/v suspension was made by isotonic phosphate buffer of 154 mM NaCl in 10 mM
Sodium Phosphate Buffer at pH 7.4 and used as stock erythrocyte or RBC suspension.
Hypotonic solution-induced haemolysis
11

The activity of the synthesized compounds was tested according to the reported method
[25]. The tested sample 0.5mL consisted of stock erythrocyte (RBC) suspension. 5 mL of
hypotonic solution (50 mM NaCl in 10 mM Sodium Phosphate buffered saline at pH 7.4) and
different concentrations of sample (20, 40, 60, 80 and 100 µg/mL) was prepared. The blank
control consisted of 0.5 mL RBC suspension mixed and 5 mL hypotonic buffered solution
alone. The mixtures were incubated for 10 minutes at room temperature, centrifuged for 10
minutes at 3000 rpm and supernatant was measured by spectrophotometrically at 540 nm.
The % inhibition of haemolysis was calculated according to the following formula.
Where:
A1 = Absorbance of hypotonic buffered solution alone
A2 = Absorbance of test /standard sample in hypotonic solution
4.4 Anti-inflammatory activity by COX-2 as model enzyme
The enzymatic in vitro assays using human recombinant COX-2 (Sigma-Aldrich)
were performed to test the inhibitory activities of the test compounds [41]. COX-2 (0.5 unit/
reaction) was added to 180 μL of the incubation mixture consisting of 100 mM Tris buffer
(pH 8.0), 5 μM porcine hematin, 18 mM L-epinephrine, and 50 μM Na2EDTA. The test
substances were then dissolved in DMSO and added (10 μL), with the mixture incubated for
5 min at room temperature. Pure DMSO was used as a blank and (S)-(+)-ibuprofen (Sigma-
Aldrich) as a reference inhibitor. Then, 5 μL of 10 μM arachidonic acid was added, and the
reaction was incubated for 20 min at 37 °C. The reaction was terminated by adding 10 μL of
10% (v/v) formic acid. Prostaglandin E2 (PGE2), the main product of the reaction, was
quantified using a Prostaglandin E2 ELISA kit (Enzo Life Sciences, NY, USA). The samples
were diluted 1:15 in assay buffer (provided in the kit), and then the incubation was performed
according to the manufacturer’s instructions. The absorbance at 405 nm was measured using
12

a Tecan Infinite M200 microplate reader (Tecan Group, Switzerland). The inhibitory activity
was expressed as the percent inhibition compared to the blank. Experiments were repeated at
least three times with two technical replicates for screening as well as to determine the IC50
values
4.5 Antioxidants activity
4.5.1 DPPH (1,1-diphenyl-2-picryl-hydrazyl) assay
The radical scavenging activities of DPPH free radicals by synthesized compounds
were determined according to the reported method [42]. Briefly, 50 µL of test compounds
was mixed at different concentrations (20, 40, 60, 80 and 100 µg/mL) with 1 mL of 0.1 mM
DPPH in methanol solution and 450 µL of 50 mM Tris HCl buffer (pH 7.4). Methanol (50
µL) only was used as the experimental control. After 30 min of incubation at room
temperature, the reduction in the number of DPPH free radicals was measured by reading the
absorbance at 517 nm. BHA, AA and GA were used as standards similar to test
concentrations. Percent inhibition was calculated from the following equation:
4.5.2 ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sufonic acid) assay
The ability of the test sample to scavenge ABTS.+ radical cation was determined
according to the literature method [43] with slight modifications. The ABTS.+ radical cation
was pregenerated by mixing 7 mM ABTS.+ stock solution with 2.45 mM potassium persulfate
(final concentration) and incubating for 12–16 hrs in the dark at room temperature until the
reaction was complete and the absorbance was stable. The absorbance of the ABTS.+ solution
was equilibrated to 0.70 (± 0.02) by diluting with distilled water at room temperature, then 2
mL was mixed with different concentration of the test sample (20, 40, 60, 80, and 100
µg/mL) and the absorbance was measured at 734 nm after 6 min. The scavenging capability
of ABTS.+ radical was calculated using the following equation:
13

ABTS.+ scavenging effect (%) = [(Ac-As) /Ac] x 100
Where, Ac is the initial concentration of the ABTS.+ and As is the absorbance of the
remaining concentration of ABTS.+ in the presence of compounds.
4.5.3 DMPD (N, N-dimethyl-p-phenylenediamine) assay
The DMPD radical scavenging ability of synthesized compounds was determined by
the method [44] with slight modifications. This assay is based on the capacity of the extract
to inhibit DMPD.+ cation radical formation. Briefly, 105 mg of DMPD was dissolved in 5 mL
of distilled water. Then, 1 mL of this solution was added to 100 mL of 0.1 M acetate buffer
(pH 5.3). DMPD.+ was produced by adding 0.3 mL ferric chloride (0.05 M) to this solution.
Different concentrations of standard antioxidants or synthesized compounds (20, 40, 60, 80
and 100 μg/mL) were added, and the total volume was adjusted to 1 mL with distilled water.
One millilitre of the DMPD.+ solution was directly added to the reaction mixture. The
reaction mixtures were incubated in the dark for 15 min. The absorbance was measured at
505 nm.
4.6 Molecular docking studies
The co-ordinates of H⁺ /K⁺ -ATPase were obtained from the Brookhaven Protein Data
Bank, whose PDB ids are 1E9Y and 2ZXE respectively. Ligands were drawn using Maestro
2D sketcher and energy minimize was computed by OPLS 2005. Proteins were prepared by
retrieving into Maestro platform (Schrödinger, Inc.). Protein structures were corrected, by
using a Prime software module of Schrödinger to correct the missing loops and in the protein.
Water molecules from H⁺ /K⁺ -ATPase were removed beyond 5Å from the hetero atom
respectively. Water molecules which are through to be important in aiding the interaction
between the receptor were optimized during protein pepwizard. Automated, necessary bonds,
bond orders, hybridization, explicit hydrogens and charges were assigned. OPLS 2005 force
14

field was applied to the protein to restrained minimization and RMSD of 0.30Å was set to
converge heavy atoms during the preprocessing of protein before starting docking. Using
Extra-precision (XP) docking and scoring each compound were docking into the receptor grid
of radii 20Å × 20Å × 20Å and the docking calculation were judged based on the Glide score
of all the ligands and molecular visualization was done under Maestro workspace [45].
Acknowledgement
We are grateful to Wuhan University of Technology, China and Rani Channamma
University, Belagavi, India for their continuous encouragement towards the research and
financial support.
References:
1.
2.
3.
C. Vergelli, M. P. Giovannoni, S. Pieretti, A. D. Giannuario, V. Dal Piaz, Bioorg.
Med. Chem. 15 (2007) 5563-5575.
T. K. Motawi, H. M. Abd Elgawad, N. N. Shahin, J. Biochem. Mol Toxicol. 21
(2007) 280-288.
Y. Nakano, E. Kuroda, T. Kito, S. Uematsu, S. Akira, A. Yokota, S. Nishizawa, U.
Yamashita, J. Neurosurg. 108 (2008) 311-319.
4.
5.
6.
7.
8.
P. Singh, A. Mittal, Mini, Rev. Med. Chem. 8 (2008) 73-90.
S. Gustavsson, H. O. Adam, L. Loof, Lancet. 8342 (1983) 124-125.
K. P. Rakesh, C. S. Shantharam, H. M. Manukumar, Bioorg. Chem. 68 (2016) 1- 8.
S. Rollas, S. G. Kucukguzel, Molecules. 12 (2007) 1910-1939.
D. H. Soliman, M. A. Abdelrahman, M. S. Gomaa, M. M. Elasser, M. M. Abdel-
Aziz, I. Salama, Eur. J. Med. Chem. 138 (2017) 698-714.
9.
C. M. Moldovan, O. Oniga, A. Pârvu, Brîndus¸ Tiperciuc, P. Verite, A. Pirnau, O.
Crisane, M. Bojita, R. Pop, Eur. J. Med. Chem. 46 (2011) 526-534
10. H. S. Kareem, A. Ariffin, N. Nordin, T. Heidelberg, A. Abdul-Aziz, K. W. Kong,
W. A. Yehye, Eur. J. Med. Chem. 103 (2015) 497-505.
15

11. P. Hernandez, M. Cabrera, M. L. Lavaggi, L. Celano, I. Tiscornia, T. R. D. Costa,
L. Thomson, M. B. Fogolin, A. L. P. Miranda, L. M. Lima, E. J. Barreiro, M.
Gonzalez, H. Cerecetto, Bioorg. Med. Chem. 320 (2012) 2158-2171.
12. S. Carradori, D. Secci, A. Bolasco, D. Rivanera, E. Mari, A. Zicari, L.V. Lotti, B.
Bizzarri, Eur. J. Med. Chem. 65 (2013) 102-111.
13. E. Vavrikova, S. Polanc, M. Kocevar, K. Horvati, S. Bosze, J. Stolarikova, K.
Vavrova, J. Vinsova, Eur. J. Med. Chem. 46 (2011) 4937-4945.
14. P. Nagender, R. Naresh Kumar, G. Malla Reddy, D. Krishna Swaroop, Y.
Poornachandra, C. Ganesh Kumar, B. Narsaiah, Bioorg. Med. Chem. Lett. 26
(2016) 4427-4432.
15. Zhong-Hua Li, Dong-Xiao Yang, Peng-Fei Geng, Ji Zhang, Hao-Ming Wei et al.,
Eur. J. Med. Chem. 124 (2016) 967-980.
16. Guangcheng Wang, Ming Chen, Jing Wang, Yaping Peng, Luyao Li, ZhenZhen
Xie, Bing Deng, Shan Chen, Wenbiao Li, Bioorg. Med. Chem. Lett. 27 (2017)
2957-2961.
17. A. R. Todeschini, A. L. P. Miranda, K. C. Silva, S. C. Parrini, E. J. Barreiro, Eur. J.
Med. Chem. 33 (1998) 189-199.
18. J. R. Dimmock, S. C. Vashishtha, J. P. Stables, Eur. J. Med. Chem. 35 (2000) 241-
248.
19. Gao-Feng Zha, Jing Leng, N. Darshini, T. Shubhavathi, H. K. Vivek, A. M. Asiri,
H. M. Marwani , K. P. Rakesh, N. Mallesha, Hua-Li Qin, Bioorg. Med. Chem.
Lett. 27 (2017) 3148-3155.
20. Shi-Meng Wang, Gao-Feng Zha, K. P. Rakesh, N. Darshini, T. Shubhavathi, H. K.
Vivek, N. Mallesha, Hua-Li Qin, Med. Chem. Commun. 8 (2017) 1173-1189
21. K. P. Rakesh, A. B. Ramesha, C. S. Shantharam, K, Mantelingu, N. Mallesha, RSC
Adv. 6 (2016) 108315-108318.
16

22. K. P. Rakesh, N. Darshini, S. L. Vidhya, Rajesha, N. Mallesha, Med. Chem. Res.
26 (2017) 1675-1681.
23. Xing Chen, Jing Leng, K.P. Rakesh, N. Darshini, T. Shubhavathi, H.K. Vivek, N.
Mallesha, Hua-Li Qin, Med. Chem. Comm. 8 (2017) 1706-1719.
24. A. Sharma, R. Suhas, K. V. Chandana, S. H. Banu, D. C. Gowda, Bioorg. Med.
Chem. Lett. 14 (2013) 4096-4098.
25. U. A. Shinde, A. S. Phadke, A. M. Nair, A. A. Mungantiwar, V. J. Dikshit, M. N.
Saraf, Fitoterapia. 70 (1999) 251-257.
26. D. M. Lokeshwari, D. K. Achutha, B. Srinivasan, S. Naveen, N. K. Lokanath, K.
Ajay Kumar, Bioorg. Med. Chem. Lett. 27 (2017) 3806-3811.
27. A. G. Lafuente, E. Guillamon, A. Villares, M. A. Rostagno, J. A. Martinez,
Inflamma. Res. 58 (2009) 537-552.
28. H. P. Kim, K. H. Son, H. W. Chang, S. S. Kang, S. S. J. Pharmaco. Sci. 96 (2004)
229-245.
29. A. L. Blobaum, L. J. Marnett, J. Med. Chem. 50 (2007) 1425-1441.
30. K. Seibert, Y. Zhang, K. Leahy et al., Proc. Natl. Acad. Sci. U S A. 91 (1994)
12013-12017.
31. H. L. Willingale, N. J. Gardiner, N. McLymont, S. Giblett, B. D. Grubb, Br. J.
Pharmacol. 122 (1997) 1593-1604.
32. L. P. Leong, G. Shui, Food. Chem. 76 (2002) 69-75.
33. Y. L. Lee, M. T. Yen, J. L. Mau, Food. Chem. 104 (2007) 1-9.
34. K. P. Rakesh, H. M. Manukumar, D. C. Gowda, Bioorg. Med. Chem. Lett. 25
(2015) 1072-1077.
35. A. Husain, M. Ajmal, Acta. Pharm. 59 (2009) 223-233.
36. S. P. Sharma, N. Kumar, R. Dudhe, Der. Pharm. Chem. 2 (2010) 253-263.
17

37. O. H. Lowry, N. J. Rosebrough, A. L. Farr, R. J. Randall, J. Biol. Chem. 193
(1951) 265-275.
38. C. H. Fiske, Y. Subbarow, J. Biol. Chem. 66 (1952) 375-400.
39. W. B. Im, J. C. Sih, D. P. Blakeman, J. P. McGrath, J. Biol. Chem. 260 (1985)
4591-4597.
40. D. M. Lokeshwari, N. D. Rekha, B. Srinivasan, H. K. Vivek, K. Ajay Kumar,
Bioorg. Med. Chem. Lett, 27 (2017) 3048-3054.
41. C. Fernandes, A. Palmeira, I. I. Ramos, C. Carneiro, C. Afonso, M. E. Tiritan, H.
et al. Pharmaceuticals. 10 (2017) 50.
42. M. S. Blois, Nature. 181 (1958) 1199-1200.
43. R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang, C. Rice-Evans, Free.
Radical. Bio. Med. 26 (1999) 1231-1237.
44. V. Fogliano, V. Verde, G. Randazzo, A. Ritieni, J. Agric. Food Chem. 47 (1999)
1035-1040.
45. R. A. Friesner, J. L. Banks, R. B. Murphy, T. A. Halgren, J. J. Klicic, D. T. Mainz,
M. P. Repasky et al., J. Med. Chem. 47 (2004) 1739-1749.
18

Table 1: Biological activities of the synthesized hydrazones (1-36)
H+/K+
Anti-
Antioxidant activitya
ATPase inflammatory
COX-2
(IC50 g/mL)
Entry
activitya
IC50
activitya
IC50 [μM]a
IC50 g/mL
DPPH
ABTS
DMPD
g/mL
-
1
-
-
92±0.26
-
-
-
-
-
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
-
86±0.93
90±0.75
78±0.18
80±0.32
66±0.34
70±0.18
74±0.36
30±0.59
34±0.28
78±0.30
70±0.14
74±0.19
78±0.52
32±0.51
36±0.11
20±0.11
18±0.18
40±0.33
18±0.32
78±0.33
82±0.14
80±0.33
76±0.21
12±0.29
10±0.39
-
-
-
-
-
90±0.63
80±0.16
78±0.21
70±0.36
72±0.38
76±0.26
28±0.32
34±0.56
80±0.32
74±0.30
76±0.31
80±0.27
30±0.17
36±0.74
20±0.36
22±0.27
42±0.21
20±0.17
82±0.23
78±0.64
72±0.28
70±0.21
08±0.26
12±0.29
-
88±0.95
82±0.93
74±0.36
68±0.28
66±0.31
72±0.21
26±0.15
36±0.49
78±0.55
72±0.20
68±0.11
74±0.55
32±0.22
38±0.21
16±0.33
24±0.36
38±0.33
16±0.37
86±0.30
84±0.31
76±0.31
78±0.31
10±0.23
14±0.29
-
-
88±1.02
68±1.04
72±0.95
66±0.40
70±0.68
22±0.62
26±0.91
72±0.65
74±0.63
70±0.92
74±0.43
24±0.44
28±0.63
12±0.38
14±0.47
26±0.54
16±0.41
80±0.36
84±0.63
78±0.56
80±0.49
06±0.68
08±0.43
-
90±0.46
28±0.65
24±0.61
28±0.64
26±0.45
72±0.42
78±0.12
30±0.74
28±0.40
32±0.24
30±0.55
80±0.23
78±0.36
60±0.23
66±0.86
70±0.257
62±0.27
08±0.47
12±0.47
10±0.51
14±0.84
56±0.74
60±0.48
-
16±0.72
20±0.72
18±0.31
15±0.88
31±0.99
-
13±0.17
22±0.79
17±0.33
12±0.29
30±0.77
-
-
-
-
19±0.67
06±0.10
10±0.75
08±0.51
06±0.73
17±0.37
21±0.88
26
27
-
28
-
-
-
-
-
-
29
-
-
-
-
-
-
30
31
32
33
34
52±0.36
50±0.69
42±0.24
48±0.62
52±0.85
54±0.62
44±0.19
48±0.12
-
40±0.52
48±0.62
38±0.54
42±0.61
50±0.62
48±0.41
56±0.72
27±0.18
48±0.21
40±0.21
38±0.36
56±0.31
54±031
58±0.31
52±0.21
-
44±0.31
48±0.31
42±0.36
60±0.47
58±0.30
54±0.11
58±0.21
-
52±0.21
46±0.18
40±0.36
56±0.61
62±0.33
60±0.33
50±0.91
-
-
17±0.37
22±0.82
20±0.81
35
36
-
25±0.52
-
-
Omerazole
Indomethacin
Ibuprofen
Ascorbic acid
Gallic acid
-
-
-
44±0.15
-
-
-
-
-
-
4±0.10
-
-
44±0.19
48±0.26
48±0.26
42±0.36
42±0.69
46±0.28
a Values are mean of three determinations, the ranges of which are <5% of the mean in all cases.
19

Scheme 1: Synthesis of target compounds 4-36
20

Figure 1: Synthetic strategy for the target compounds 4-36
Figure 2: (A) Molecular interaction of H+/K+ ATPase enzyme (1E9Y) with Ligand 25 (B) Electrostatic surface representation of the
protein depicting the best-docked pose for Ligand 25.
21

Figure 3: (A) Molecular interaction of H+/K+ ATPase enzyme (2ZXE) with Ligand 25 (B) Electrostatic surface representation of the
protein depicting the best-docked pose for Ligand 25.
22

Research Highlights
1. Synthesis of 32 dihydrazones and evaluated for in vitro biological activities.
2. Compound 25 was found to be most potent H+/K+-ATPase and antioxidants activity.
3. Compound 21 showed excellent anti-inflammatory and COX-2 activities.
4. The SAR showed that, electron donating groups (OH, OCH3) favored the H+/K+-ATPase and antioxidants activity, whereas,
electron withdrawing groups (F, Cl, Br and NO2) favored the anti-inflammatory and COX-2 activity.
23

Graphical abstract
Multi-targeted dihydrazones as potent biotherapeutics
Chen Li, M. B. Sridhara, K. P. Rakesh, H. K. Vivek, H. M. Manukumar, C. S. Shantharam and Hua-Li Qin
24