Extending the scope of amantadine drug by incorporation of phenolic azo Schiff bases as potent selective inhibitors of carbonic anhydrase II, drug-likeness and binding analysis

Article abstract of DOI:10.1111/cbdd.13335

A series of Amantadine-based azo Schiff base dyes 6a–6e have been synthesized and characterized by ¹H NMR and ¹³C NMR and evaluated for their in vitro carbonic anhydrase II inhibition activity and antioxidant activity. All of the syn

Full text of DOI:10.1111/cbdd.13335

DR AAMER SAEED (Orcid ID : 0000-0002-7112-9296)
MR. FAYAZ ALI LARIK (Orcid ID : 0000-0002-8491-5229)
Article type : Research Article
Extending the scope of amantadine drug by incorporation of phenolic azo Schiff bases as
potent selective inhibitors of carbonic anhydrase II, drug likeness and binding analysis
Pervaiz Ali Channara Aamer Saeed*a, Danish Shahzada, Fayaz Ali Larik*a, Mubashir
Hassanb, Hussain Razab, Qamar Abbasc, Sung-Yum Seob
a Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan
bDepartment of Biological Sciences, College of Natural Sciences, Kongju National University,
56 Gongjudehak-Ro, Gongju, Chungnam 32588, Republic of Korea
cDepartment of Physiology, University of Sindh, Jamshoro-76080, Pakistan

Email: aamersaeed@yahoo.com, fayazali@chem.qau.edu.pk (F.A.L) Tel +92-51-9064-2128;
Fax: +92-51-9064-2241
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.13335
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Abstract
A series of Amantadine based azo Schiff base dyes 6a-6e have been synthesized and
1
13
characterized by H NMR and C NMR and evaluated for their in vitro carbonic anhydrase II
inhibition activity and antioxidant activity. All of the synthesized showed excellent carbonic
inhibition. Compound 6b was found to be the most potent derivative in the series, the IC50 of 6b
was found to be 0.0849 ± 0.00245µM (standard Acetazolamide IC50=0.9975±0.049µM). The
binding interactions of the most active analogs were confirmed through molecular docking
studies. Docking studies showed 6b is interacting by making two hydrogen bonds w at His93 and
Ser1 residues respectively. All compounds showed a good drug score and followed Lipinski’s
rule. In summary, our studies have shown that these amantadine derived phenolic azo Schiff base
derivatives are a new class of carbonic anhydrase II inhibitors.
Keywords: Amantadine Drug derivatives; phenolic azo Schiff bases; Carbonic anhydrase II
inhibition; antioxidant; Drug Score; Drug likeness; Binding analysis
1. Introduction
1-Adamantylamine is commercially known as amantadine and it contains adamantine as a
backbone and has one amine group along with four methylene groups [1]. Amantadine is an
antiviral and antiparkinsonian drug and particularly inhibits influenza A virus [2]. Moreover,
Amantadine also blocks calcium channel protein Kcv and also it assists in releasing of dopamine
[3, 4]. Apart from being the medicinally significant drug, amantadine serves as the key precursor
for incorporation of the adamantine skeleton inorganic compounds [5].
The carbonic anhydrase enzyme represents a broad class of metallo-enzymes and widely
distributed among mammals [6]. There are 15 isoforms of carbonic anhydrase are known and this
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enzyme catalyzes the cleavage of carbon dioxide [7]. The isoforms of carbonic anhydrase are
categorized into two categories i) systolic (CA I, CA II, CA III, CA VII and CA XIII) ii)
Membrane-bound (CA IV, CA IX, CA XII, and CA XIV). Carbonic anhydrase (CA, EC 4.2.1.1)
[8]
inhibitors (CAIs) for isoforms I-II (glaucoma associated) and IX-XII (cancer-associated)
.
Carbonic anhydrase (CA) plays an important role in the regulation of respiration, bone
resorption, pH regulation and CO2 homeostasis, tumorigenicity and these features ignite
medicinal chemists to search efficient and safe carbonic anhydrase inhibitors (CAI) since the
1950’s[9, 10]. Carbonic anhydrase contains Zn+2 ion as Lewis acid which assists in binding of the
active substrate with the water molecule. Zn+2 is tightly bound with three histidine units [11]. The
nucleophilic oxygen of water molecule attacks the electrophilic carbon of carbon dioxide to form
the complex with zinc ions which then collapse to release bicarbonate ions (Figure 1).
<<Insert Figure 1>>
A large number of sulfonamide based carbonic anhydrase inhibitor (CAI) drugs like are in
clinical use for more than 70 years as diuretics, antiglaucoma, anticonvulsant or anti-infective
drugs examples of clinically used CAIs: acetazolamide AZM, methazolamide MZA,
ethoxzolamide EZA, dorzolamide DZA, brinzolamide BRZ, zonisamide ZNS, sulpiride SLP,
indisulam IND, celecoxib CLX (Figure 2)[12-14]
.
<<Insert Figure2>>
However, due to numerous isoform of carbonic anhydrase enzyme, the search to obtain effective
safe drug candidates is still work in progress.
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Schiff bases possess the broad spectrum of biological activities antibacterial, antifungal, anti-
mouse hepatitis virus (MHV), inhibition of herpes simplex virus type 1 (HSV-1) and adenovirus
type 5 (Ad 5), anticancer, anti-mosquito larvae and herbicidal activities [15-17]. Moreover Schiff
base is important precursors in the synthesis of β-lactams and metal complexes [18]. It is assumed
that the azomethine moiety in Schiff bases is responsible for biochemical interactions. Azo
compounds possess academic and industrial important and are considered as building blocks in
the synthesis of metal complexes and polymers [19, 20]. Moreover, azo compounds possess
biological importance such as inhibition of DNA, RNA, and protein synthesis, nitrogen fixation,
and carcinogenesis [21]
.
Hybrid pharmacophore approach has been considered as the hallmark in seeking potent
derivatives in chemical bio drug designing. In this account, we have a hybrid pharmacophore
based strategy to design potent carbonic anhydrase (CA II) inhibitors). Amantadine drug was
chosen as core skeleton and it was linked with azo and Schiff base moieties to incorporate
intriguing structural features. Schiff bases contain imine moiety and are known as masked
carbonyl analogue of either aldehyde or ketone. The azo group exhibits diverse applications in
material chemistry has been induced as linked with amantadine drug. The hydroxyphenyl ring
was suitably substituted with various electronic perturbing groups in order to investigate the role
of substituents attached to the aryl ring. Herein, we have chosen free amino-containing
amantadine drug to extend it’s by incorporating phenolic azo Schiff bases and the synthesized
derivatives were screened against carbonic anhydrase II enzyme inhibition and using molecular
simulation drug score, drug-likeness and binding analysis were explored.
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2. Experimental
2.1 Methods and Materials
The Rf values were determined using aluminum precoated silica gel plates Kiesel 60 F254 from
Merck (Germany). Melting points of the compounds were measured in open capillaries using
Stuart melting point apparatus (SMP3) and are uncorrected. 1H-NMR spectra were determined as
13
DMSO, CDCl3 solutions at 300 MHz using a Bruker AM-300 spectrophotometer, and the C-
NMR spectra were determined at 75 MHz using a Bruker 75 MHz NMR in DMSO-d6, and
CDCl3 solutions. The elemental analysis was performed on Leco CHNS-932 Elemental Analyzer
(Leco Corporation, USA). For the synthesis of compounds, all chemicals were commercially
obtained and used without additional purification.
2.2 General procedure for synthesis of phenolic azo dyes (4a-3e) and their condensation with
Amantadine hydrochloride (5)
Suitably substituted anilines (1a-1e) (0.01mol) were dissolved in 20ml water and 3.5ml
concentrated HCl, with stirring maintaining the temperature at 0-5oC. A solution of NaNO3
(0.01ml) in 10ml water was added promptly to the solution of aniline with continuous and
vigorous stirring. Stirring was further continued for 1h maintaining the temperature in the same
range. After 1h the reaction mixture was checked for the completeness of reaction on a paper
chromatogram using water as mobile phase. The dried chromatogram was sprayed with the
solution of p-N, N-dimethyl amino benzaldehyde in ethanol as spraying agent. On completion of
the reaction, the diazonium salts (2a-2e) were kept in the freezer.
Salicylaldehyde (0.01ml) was dissolved in water (15ml) and K2CO3 (1.5g), kept in an ice bath at
temperature 0-5oC with stirring. The diazo solution was added dropwise to the stirred solution of
salicylaldehyde during 45 minutes, maintaining the pH above 8. The progress of the reaction was
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monitored by paper chromatography using H-acid solution in alkaline media. On completion, the
solids were filtered, dried in the oven at 70oC, for 3hs to afford the 4-(Benzeneazo)
salicylaldehyde derivatives (4a-4e) in 85-92% yields. In the case where regioisomeric products
were obtained; column chromatography was used for separation. Then 0.01mol (0.5g)
amantadine hydrochloride 5 was slowly added to a solution of 0.01 mol of 4-(Benzeneazo)
salicylaldehyde derivatives 0.002mol (4a-4e). After refluxing the reaction mixture for 3h, the
precipitate was cooled and collected by filtration. The precipitate was washed several times using
ethanol, followed by crystallization in ethanol and drying at 50 oC overnight. Yields > 70%.
2-((E)-((3s,5s,7s)-Adamantan-1-ylimino)methyl)-4-((E)-(4-chlorophenyl)diazenyl)phenol
(6a)
1
Dark yellow solid, M.P=240oC, Yield 88%, Rf= 0.50 (Chloroform: Ethanol 9:1), H NMR
(CDCl3, 300 MHZ); δ (ppm) 11.29 (s, 1H, Ar-OH), 9.61 (s, 1H, =N-CH), 8.14 (d,1H, Ar-H, J=
2.4Hz), 8.15 (d, 1H, Ar-H, J= 1.97Hz), 8.08 (dd, 1H, Ar-H, J=8.4, J=2.3Hz), 7.89-7.51 (m, 4H,
13
Ar-H), 7.17 (d, 1H, Ar-H, J=7.4Hz), 2.06 (m, 3H), 1.71 (m, 6H), 1.60 (m, 6H), C NMR (75
MHz CDCl3) δ (ppm), 13C NMR (75 MHz DMSO-d6) δ (ppm), 188.05 (OH-C), 163.74, 151.88,
150.46, 145.15, 130.21, 127.14, 124.33, 123.09, 122.39, 118.89, 65.5, 51.47, 35.48, 28.75;
found: C, 70.11; H, 6.09; N, 10.6.
4-((E)-(3-((E)-((3s,5s,7s)-Adamantan-1-ylimino)methyl) 4-
hydroxyphenyl)diazenyl)benzenesulfonamide (6b)
Dark orange crystalline solid, M.P=240oC, Yield 88%, Rf= 0.70 (Chloroform: Ethanol 9:1),1H
NMR (DMSO-d6, 300 MHZ); δ (ppm) 11.52 (s, 1H, Ar-OH), 10.35 (s, 1H, =N-CH), 8.18 (d,1H,
Ar-H, J= 2.5Hz), 8.11 (dd, 1H, Ar-H, J=8.7, J=2.4Hz), 7.80 (m, 4H, Ar-H), 7.53 (s, 4H), 7.20 (d,
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1H, Ar-H, J=7.5Hz), 2.07 (m, 3H), 1.74 (m, 6H), 1.59 (m, 6H), 13C NMR (75 MHz DMSO-d6) δ
(ppm), 13C NMR (75 MHz DMSO-d6) δ (ppm), 190.05 (OH-C), 162.84, 151.91, 150.94, 145.21,
130.23, 127.16, 124.31, 123.09, 122.39, 118.89, 65.52, 51.47, 35.48, 28.75, Anal.Calcd. for
C23H26N4O3S: C, 62.99; H, 5.98; N, 12.78; S, 7.31; found: C, 62.99; H, 5.98; N, 12.78; S, 7.31
Sodium-4-((E)-(3-((E)-((3s,5s,7s)-adamantan-1-ylimino)methyl)-4
hydroxyphenyl)diazenyl)benzenesulfonate (6c)
1
Dark yellow crystalline solid, M.P=240oC, Yield 88%, Rf= 0.60 (Chloroform: Ethanol 9:1), H
NMR (DMSO-d6, 300 MHZ); δ (ppm) 11.54 (s, 1H, Ar-OH), 10.37 (s, 1H, =N-CH), 8.19 (d,1H,
Ar-H, J= 2.5Hz), 8.11 (dd, 1H, Ar-H, J=8.7, J=2.4Hz), 7.80 (m, 4H, Ar-H), 7.21 (d, 1H, Ar-H,
13
J=7.5Hz), 2.08 (m, 3H), 1.74 (m, 6H), 1.59 (m, 6H), C NMR (75 MHz DMSO-d6) δ (ppm),
191.05 (OH-C), 163.84, 151.99, 150.94, 145.29, 130.26, 127.16, 124.33, 123.09, 122.39, 118.89,
65.53, 51.47, 35.48, 28.75, Anal.Calcd. for C23H24N3NaO4S: C, 59.86; H, 5.24; N, 9.10; S, 6.95
found: C, 59.81; H, 5.19; N, 9.02; S, 6.89.
2-((E)-((3s,5s,7s)-Adamantan-1-ylimino)methyl)-4-((E)-p-tolyldiazenyl)phenol (6d)
Light yellow solid, M.P=240oC, Yield 88%, Rf= 0.70 (n-Hexane: Ethyl acetate 6:4), 1H NMR
(CDCl3, 300 MHZ); δ (ppm) 11.21 (s, 1H, Ar-OH), 9.49 (s, 1H, =N-CH), 8.14 (d,1H, Ar-H, J=
2.4Hz), 8.12 (d, 1H, Ar-H, J= 1.97Hz), 8.05 (dd, 1H, Ar-H, J=8.4, J=2.3Hz), 7.93, (dd, 4H, Ar-
H, J=8.8Hz), 7.14 (d, 1H, Ar-H, J=7.4Hz), 2.34(s, 3H), 2.06 (m, 3H), 1.71 (m, 6H), 1.60 (m,
13
6H), C NMR (75 MHz CDCl3) δ (ppm), 186.05 (OH-C), 159.84, 151.99, 150.94, 145.22,
130.26, 127.16, 124.23, 123.03, 122.31, 118.89, 65.43, 51.44, 35.48, 28.75, 21.86, Anal.Calcd.
for C24H27N3O: C, 77.18; H, 7.29; N, 11.25: found: C, 77.12; H, 7.26; N, 11.21.
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2-((E)-((3s,5s,7s)-Adamantan-1-ylimino)methyl)-4-((E)-((3-
chlorophenyl)imino)methyl)phenol (6e)
1
Dark yellow solid, M.P=240oC, Yield 88%, Rf= 0.50 (n-Hexane: Ethyl acetate 6:4), H NMR
(CDCl3, 300 MHZ); δ (ppm) 11.30 (s, 1H, Ar-OH), 9.63 (s, 1H, =N-CH), 8.14 (d,1H, Ar-H, J=
2.4Hz), 8.16 (d, 1H, Ar-H, J= 1.97Hz), 8.08 (dd, 1H, Ar-H, J=8.4, J=2.3Hz), 8.14-7.52 (m, 4H,
13
Ar-H), 7.19 (d, 1H, Ar-H, J=7.4Hz), 2.07 (m, 3H), 1.73 (m, 6H), 1.60 (m, 6H), C NMR (75
MHz CDCl3) δ (ppm), 187.01 (OH-C), 163.68, 151.77, 150.34, 145.07, 130.22, 128.08, 129.06,
127.10, 124.33, 123.09, 122.39, 118.89, 65.5, 51.47, 35.48, 28.7 Anal.Calcd. for C24H25ClN2O:
C, 73.36; H, 6.41; N, 7.13; found: C, 73.30; H, 6.36; N, 7.09.
2.3 Carbonic anhydrase assay
Carbonic anhydrase inhibition was measured as described previously with some modifications
[28]. The method is based on the principle that p-nitrophenyl acetate is hydrolyzed by Carbonic
anhydrase to form yellow colored p-nitrophenol which was measured spectrophotometrically.
Briefly, the Reaction mixture contained 120 µL of 50 mM Tris-sulfate buffer (pH 7.6 containing
0.1 mM ZnCl2), 20 µL of inhibitor and 20 µL (50 U) bovine enzyme per well. Contents were
well mixed and pre-incubated at 25 °C for 10 min. substrate p-nitrophenyl acetate was prepared
(6 mM stock using <5% acetonitrile in the buffer and used fresh every time) and 40 µL was
added per well to achieve 0.6 mM concentration per well. The total reaction volume was made to
200 µL. After 30 min incubation at 25 °C contents was mixed and absorbance was measured at
348 nm using a microplate reader. Acetazolamide was used as a reference inhibitor and Tris-
sulfate buffer was used as negative control. Each concentration was analyzed in three
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independent experiments. IC50 values were calculated by nonlinear regression using GraphPad
Prism 5.0.
Inhibition (%)
Here, the B and S are the absorbances for the blank and samples.
2.4 Repossession of Carbonic anhydrase II from PDB
The three dimensional (3D) crystal structure of carbonic anhydrase II was retrieved from the
Protein Data Bank (PDB) having PDBID 1V9E (www.rcsb.org). Energy minimization of target
structure was carried out by using conjugate gradient algorithm and Amber force field in UCSF
Chimera 1.10.130. The stereo-chemical properties, Ramachandran graph, and values of Carbonic
anhydrase II structure were assessed by Molprobity server, while the hydrophobicity graph was
generated by Discovery Studio 4.1 Client [31-33]. The protein architecture and statistical
percentage values of helices, beta-sheets, coils, and turns were accessed by using online tool
VADAR 1.834.
2.5 In-silico designing of synthesized compounds
The synthesized ligand molecules (6a-e) were sketched in drawing ACD/ChemSketch tool and
further minimized by visualizing software UCSF Chimera 1.10.1. The different online drug
assessment tools like Molinspiration (http://www.molinspiration.com/) and Molsoft
(http://www.molsoft.com/) were employed to predict the drug-likeness and biological properties
of these designed candidate molecules. The number of rotatable bonds, hydrogen bond acceptors
(HBA) and hydrogen bond donors (HBD) were also confirmed by PubChem
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(https://pubchem.ncbi.nlm.nih.gov/). Moreover, Lipinski’s rule of five was analyzed using
Molsoft and Molinspiraion tools.
2.6 Molecular docking of synthesized compounds using AutoDock
Molecular docking experiment study was done for all the synthesized ligands (6a-e) against
urease using diverse AutoDock 4.2 tool according to the specified instructions35. In receptor
(Carbonic anhydrase-II) the polar hydrogen atoms and Kollman charges were apportioned and
for the ligand, Gasteiger partial charges were designated and non-polar hydrogen atoms were
merged. All the torsion angles for all the synthesized ligands were set free to rotate through
docking experiments. The docking experiments were performed by considering receptor as a
rigid whereas ligands as flexible molecules. A grid map values were adjusted as of 80 Å x 80 Å x
80 Å on the targeted protein to generate the grid map (Figure S1). The number of docking runs
for each docking experiment was set to 100 for the best conformational state. The Lamarckian
genetic algorithm (LGA) and empirical free energy function were applied by taking docking
parameters default. The docked complexes were further evaluated on lowest binding energy
(Kcal/mol) values and hydrogen/hydrophobic bond analysis to choose the best docking pose
using Discovery Studio (4.1) and UCSF Chimera 1.10.1.
3. Results and Discussion
3.1 Chemistry
The synthesis of amantadine drug derivatives has been outlined in scheme 1. Suitable substituted
aromatic anilines were used precursors and the amine functionality of aniline was reduced by
using method already reported in the literature. The diazonium salt formed after the reduction of
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anilines was coupled with salicylaldehyde to construct azo linkage (4a-4e). The aldehyde
functionality was utilized to obtain Schiff bases with amantadine as core nucleus 6a-6e.
Scheme 1. Synthetic route towards salicylaldehyde based azo dyes and their condensation with
adamantine (6a-6e)
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The synthesized compounds were characterized through spectroscopic and elemental analysis.
1H-NMR spectra revealed the formation of compounds and tentative signal assignments
confirmed the newly constructed linkages. The characteristic imine proton appeared at around
8.5-9.5ppm value. The appearance of signals at 7-8 ppm value was attributed with the phenyl
ring. The sp3 carbons of amantadine show a slight variation in chemical environment and were
observed at around 2-3 ppm value. 13C-NMR spectra showed the presence of an isochronous
carbons in compounds. The carbon of imine moiety appeared at around 165-170 ppm value. The
appearance of signals at around 120-140 ppm were attributed to aryl ring and sp3 carbons
appeared at 18-28 ppm value (Figure S2 and S3).
3.2 Carbonic anhydrase II assay
The results of carbonic anhydrase enzyme inhibition activity are summarized in Table 1. All the
synthesized derivatives 6a-6e exhibited CA II inhibition in micromolar range and better than the
standard used (Acetazolamide 0.9975±0.0489µM) except 6d. Compound 6d showed poor
activity compared to other derivatives. 6d contains a methyl group at the para position of the
phenyl ring and methyl group exerts electron donating effect through hyperconjugation. The
derivative 6b and 6e were found to be more potent compared to other derivatives in the series.
Table 1. Carbonic anhydrase activity (IC50 µM) of compounds (6a-6e)
6a
0.20 ± 0.01
6b
0.084 ± 0.002
0.144 ± 0.006
6.64 ± 0.33
6c
6d
6e
0.043 ± 0.002
0.99 ± 0.04
Acetazolamide
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For calculation of IC50, six to eight concentrations were used. IC50 values were calculated by
nonlinear regression using GraphPad Prism 5.0.
The higher potency of compound 6b can be rationalized as depicted in Figure 3. The amino
group of sulfonamide linked at the para position of phenyl ring forms the tetrahedral complex
with the active site of carbonic anhydrase enzyme.
<<Insert Figure 3>>
Compound 6e showed most significant carbonic anhydrase inhibition and it bears chlorine atom
at meta position of phenyl ring which is involved in the charge separation through negative
inductive effect. The aryl ring was tailored with a hydroxyl group which resulted in enhancement
of inhibition by showing interactions with histidine residue of carbonic anhydrase enzyme
(Figure 4).
<<Insert Figure 4>>
The overall carbonic anhydrase inhibition activity order was found to be in the following order
m-chloro> p-SO2NH2>SO3Na>p-chloro>p-methyl
3.3 Chemo-informatics properties and Lipinski Rule (RO5) evaluation of ligands
The designed ligands were analyzed computationally to predict the best ligand on the basis of
chemical and bio-molecular properties and RO5. The predicted chemo-informatics properties
like LogP, HBD, HBA, molar volume, polar surface area (PSA) and drug-likeness values of
ligand molecules are mentioned in Table 2. It has been confirmed from previous research data
that the standard values for molecular weight (MW) and polar surface area (PSA) are (160 to 480
g/mol) and (<89 Å2) respectively [22, 23]. The predicted results of compounds (6a-e) showed
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good, MW and PSA values which are comparable with standard values. RO5 also confirmed the
therapeutic potential of all the ligands. Hydrogen-bonding capacity has been identified as an
important parameter for describing drug permeability. Research data revealed poor permeation is
more likely to be observed when the HBA and HBD are exceeded than 10 and 5 respectively
[24]. The chemo-informatics analysis justified that all the designed compounds possess <10
HBA and <5 HBD. Moreover, their logP values were also comparable with the standard value.
However, there are plenty of examples available for RO5 violation amongst the existing drugs
[25, 26]. The predicted chemo-informatics values of all the designed ligand are mentioned in
Table 2.
Table 2. Chemo-informatics analysis of designed chemical compounds
PSA Mol.Vol
Mol.
No.
No.
Mol.
Drug
Score
Ligands
(A2) (cm3)
Wt(g/mol) HBA HBD Logp(mg/L)
6a
6b
6c
6d
6e
395.18
440.19
460.50
375.23
395.18
2
5
4
2
2
3
5
5
3
3
5.70
3.81
3.70
5.40
5.71
48.77 377.75
99.37 407.55
90.37 405.50
48.77 381.50
48.77 377.83
-0.02
-0.34
-0.32
-0.58
-0.20
Abbrevation: HBA= No of hydrogen bond acceptor, HBD= No of hydrogen bond donor, LogP=
lippophilicity of partition coefficient, LogS= lippopilicity of water, PSA= polar surface area,
MR=Molar refractivity, PZ=polarizability
3.4 Molecular docking and binding energy analysis
The docked complexes of all the compounds 6a-e against carbonic anhydrase II were analyzed
separately and evaluated on the basis of minimum energy values and ligand interactions pattern.
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Results showed that all compounds 6a-e showed good binding energy value -4.62, -5.04, -5.21, -
4.65 and -5.44 kcal/mol, respectively and exhibited in the active region of the target protein
(Table. 3). Prior research showed that the standard error for Autodock is testified as 2.5 kcal/mol.
However, in all docking complexes, the predicted energy values difference was less than
standard energy value. Although, the basic nucleus of all the synthesized compounds was
similar, therefore most of the ligands possess good efficient energy values and have no big
energy fluctuations difference. The comparative docking analysis and inhibition constant (IC50)
value justified that 6b has good therapeutic potential as compared to all other compounds.
Table 3. Docking results of synthesized compounds using AutoDock
Electrostatic Torsional
Binding
Ligands energy
(kcal/mol)
Inhibition Intermoleculr
Ligand
efficiency
energy
(kcal/mol)
-0.16
energy
(kcal/mol)
2.39
constant
(uM)
energy
(kcal/mol)
6a
6b
6c
6d
6e
-4.62
-5.04
-5.21
-4.65
-5.44
-0.13
-0.16
-0.19
-0.17
-0.19
270.21
202.98
150.85
388.95
102.69
-6.01
-7.13
-6.70
-6.14
-6.93
0.02
2.09
0.10
1.49
0.01
1.49
-0.07
1.49
3.5 Binding analyses of synthesized compounds against carbonic anhydrase II
The ligands-protein binding analyses showed that 6b confined in the active binding pocket of the
target protein as mentioned in Figure 5. The CA II has an active site cleft (15 Å in diameter and
15 Å deep), and contains a Zinc2+ ion that is coordinated in a tetrahedral geometry with three
histidine residues (His94, His96 and His119) and a water molecule/hydroxide ion[27]. The 6b-
receptor docked complex reveals the best conformational state with hydrogen bond interactions
within the receptor binding pocket. The docking result of 6b-receptor docked complex showed
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that two hydrogen bonds were observed at His93 and Ser1 residues respectively. The oxygen
moiety of functional group in 6b interacts with His93 with bond distance 3.51Å while OH group
on benzene ring form another hydrogen bond against Ser1 with bond length 2.06Å respectively
(Figure 5). No π-π stacking interactions were observed between ring structure of ligands and
aromatic residues. The 2D conformations and binding pose and interactions with binding
residues of all the candidate molecules are mentioned in (Figure. S4-S7)
<<Insert Figure 5>>
3. Conclusions
A large number of isoforms of carbonic anhydrase enzyme provide an incentive to design and
develop safe and effective inhibitors. The use of hybrid pharmacophore approach is one of the
key strategy in modern 4D (drug design discovery and development). In this account, we utilized
hybrid pharmacophore approach by incorporating phenolic azo Schiff bases in amantadine drug.
Azo and imine moieties were combined with amantadine drug. The synthesized derivatives 6a-6e
were subjected to carbonic anhydrase and antioxidant activity. The derivative 6b and 6e were
found to significant derivatives and showed better potential than the standard acetazolamide. The
compound 6b showed great therapeutic potential and exhibited good drug score -0.34. The
docking result of 6b-receptor docked complex showed that two hydrogen bonds were observed at His93
and Ser1 residues respectively. RO5 also confirmed the therapeutic potential of all the ligands.
Hydrogen-bonding capacity has been identified as an important parameter for describing drug
permeability. The results of carbonic anhydrase enzyme inhibition suggest that these synthesized
derivatives can serve as structural templates in drug designing and discovery.
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Conflict of Interest
Authors declare no any conflict of interest
List of figures legend, manuscript file
Figure 1. Mechanism of action of carbonic anhydrase
Figure 2. Some potent carbonic anhydrase inhibitors containing sulfonamides and sulfamates.
Figure 3. Amino group of 6b interacts with zinc ions associated with histidine units
Figure 4. Hydroxyl group showing interactions with histidine residue
Figure 5. Docking interaction 6b with the receptor molecule. A) The protein structure is represented in
grey color while the interacted residues are justified in light maroon color. B) The closer view of binding
interaction. The ligand molecule is depicted in purple color while their functional groups such as oxygen,
nitrogen and sulfur are shown in red, blue and yellow colors respectively. Amino acids are highlighted in
maroon color and purple dotted lines justify the hydrogen binding with distance mentioned in angstrom
(Å). Two hydrogen bonds were observed at His93 and Ser1 position in the target protein. Zinc metal is
represented in the grey circle.
List of figure legends of supplementary information file
Figure.S1 A) Crystal structure of bovine anhydrase II. B) Ramachandran graph accessed from
PDB.
Figure S2 13C-NMR spectrum of 6b
Figure S3 1H-NMR spectrum of 6b
Figure S4. Docking 6a against target protein
Figure S5. Docking 6c against target protein
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Figure S6. Docking 6d against target protein
Figure S7. Docking 6e against target protein
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.