Synthesis of sulfonamide, amide and amine hybrid pharmacophore, an entry of new class of carbonic anhydrase II inhibitors and evaluation of chemo-informatics and binding analysis

Article abstract of DOI:10.1016/j.bioorg.2019.01.060

Selective inhibition of carbonic anhydrase (CA) enzyme is an active area of research for medicinal chemists. In the current account, a hybrid pharmacophore approach was employed to design sulfonamide, amide and amine containing new series of potent carbonic anhydrase II inhibitors. The aromatic fragment associated with pharmacophore was altered suitably in order to find effective inhibitors of CA-II. All the derivatives 4a-4m showed better inhibition compared to the standard acetazolamide. In particular, compound 4l exhibited significant inhibition with IC₅₀ value of 0.01796 ± 0.00036 μM. The chemo-informatics analysis justified that all the designed compounds possess <10 HBA and <5 HBD. The ligands-protein binding analyses showed that 4l confined in the active binding pocket with three hydrogen bonds observed with His63, Asn66 and Thr197 residues.

Full text of DOI:10.1016/j.bioorg.2019.01.060

Accepted Manuscript
Synthesis of Sulfonamide, amide and amine hybrid pharmacophore, an entry of
new class of carbonic anhydrase II inhibitors and evaluation of chemo-infor-
matics and binding analysis
Attique Ahmed, Pervaiz Ali Channar, Aamer Saeed, Markus Kalesse, Mehar
Ali Kazi, Fayaz Ali Larik, Qamar Abbas, Mubashir Hassan, Hussain Raza,
Sung-Yum Seo
PII:
S0045-2068(18)31078-2
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.060
YBIOO 2764
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 September 2018
12 January 2019
27 January 2019
Please cite this article as: A. Ahmed, P. Ali Channar, A. Saeed, M. Kalesse, M. Ali Kazi, F. Ali Larik, Q. Abbas,
M. Hassan, H. Raza, S-Y. Seo, Synthesis of Sulfonamide, amide and amine hybrid pharmacophore, an entry of new
class of carbonic anhydrase II inhibitors and evaluation of chemo-informatics and binding analysis, Bioorganic
Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.01.060
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Synthesis of Sulfonamide, amide and amine hybrid pharmacophore, an entry of new class
of carbonic anhydrase II inhibitors and evaluation of chemo-informatics and binding
analysis
Attique Ahmeda, Pervaiz Ali Channara, Aamer Saeeda, Markus Kalesseb, Mehar Ali Kazic,
Fayaz Ali Larik*a, Qamar Abbasd, Mubashir Hassane, Hussain Razae, Sung-Yum Seoe
a Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan.
bInstitut für Organische Chemie, Schneiderberg 1 B, 30167 Hannover, Germany.
cDepartment of Physiology, University of Sindh, Jamshoro, 76080, Pakistan
dInstitute of Biochemistry, University of Sindh, Jamshoro, 76080, Pakistan
eDepartment of Biological Sciences, College of Natural Sciences, Kongju National University,
56 Gongjudehak-Ro, Gongju, Chungnam 32588, Republic of Korea
Email: aamersaeed@yahoo.com; fayazali@chem.qau.edu.pk (F.A.L) Tel +92-51-9064-2128; Fax:
+92-51-9064-2241
1

Graphical Abstract
2

Abstract
Selective inhibition of carbonic anhydrase (CA) enzyme is an active area of research for
medicinal chemists. In the current account, a hybrid pharmacophore approach was employed to
design sulfonamide, amide and amine containing new series of potent carbonic anhydrase II
inhibitors. The aromatic fragment associated with pharmacophore was altered suitably in order to
find effective inhibitors of CA-II. All the derivatives 4a-4m showed better inhibition compared
to the standard acetazolamide. In particular, compound 4l exhibited significant inhibition with
IC50 value of 0.01796±0.00036 µM. The chemo-informatics analysis justified that all the
designed compounds possess <10 HBA and <5 HBD. The ligands-protein binding analyses
showed that 4l confined in the active binding pocket with three hydrogen bonds observed with
His63, Asn66 and Thr197 residues.
Keywords: Carbonic anhydrase II inhibition; Synthesis; Hybrid Pharmacophore; Binding
analysis; Chemo-informatics
3

1. Introduction
The carbonic anhydrases (CAs, E.C. 4.2.1.1) are a group of zinc metal containing
metalloenzymes pervasive in nature. The three most prevalent isoforms in nature include, -
CAs (present in vertebrates, eubacteria, algae and cytoplasm of green plants), the -CAs
(predominantly in eubacteria, algae and chloroplasts of both mono- as well as dicotyledons) and
the -CAs (mainly in archaea and some eubacteria). In higher vertebrates including humans, the
different carbonic anhydrase isozymes include cystolic forms (CA I-III, CA VII), four membrane
bound isozymes (CA IV, CA IX, CA XII and CA XIV), one mitochondrial form (CA V) and one
secreted CA isozyme, CA VI [1-4]. The carbonic anhydrase catalyze an apparently simple
-
physiological interconversion (CO2 + H2O
HCO3 + H+) yet this simple reaction is involved
in the crucial process of respiration and transport of CO2/bicarbonate between metabolizing
tissues and lungs, pH and CO2 homeostasis, electrolyte secretion in a variety of tissues/organs,
biosynthetic reactions (such as gluconeogenesis, lipogenesis and ureagenesis), bone resorption,
calcification, and tumorigenicity [5-8]. The CA isozymes have been the active area of research
among medicinal chemists because designing of inhibitors of CA play important role in the
treatment of epilepsy, glaucoma, idiopathic intracranial hypertension and altitude sickness.
Sulfonamides possess many types of biological activities, and representatives of this class of
pharmacological agents are widely used in clinic as antibacterial, hypoglycemic, diuretic, anti-
hypertensive and antiviral drugs among others [9-15]. Sulfonamides (R-SO2NH2 possess great
significance in the designing of CA inhibitors mainly due to the unique and tailor ability of
binding with CA protein which is driven by coordination of the deprotonated sulfonamide
nitrogen to the catalytic zinc ion (Zn2+), additional interactions with the hydrophilic and/or
hydrophobic region of the active site may take place, depending on the nature of the substituent
4

group. After the first report of the scientific evidence of sulfanilamide as inhibitor of CA
enzymes [16], it was suggested that unsubstituted aromatic sulfonamides of type ArSO2NH2 act
as strong CAIs (carbonic anhydrase inhibitors) which led to the discovery of several new drug
candidates. Currently, a number of research groups are actively involved in the design and
discovery of sulfonamides as carbonic anhydrase inhibitors [17-21]. Despite substantial synthetic
efforts, it is rather difficult to achieve high isozyme selectivity in vitro with classical designs
incorporating aromatic/heterocyclic primary sulfonamides and sulfamates due to sequence
homology and thus structural similarities that exist between different CA isozymes in their active
sites.
Selective inhibition of carbonic anhydrase II constitutes a viable approach to fight against
the disorders caused by harmful effects of CA II enzyme [22]. Building on these premises and
taking into account the fact that the amide functionality also possesses the ability to interact with
active site of CA II through hydrogen bonding, besides sulfonamide, and incorporation of amine
in the target compounds can further enhance their aptitude to bind effectively. We herein report a
hybrid pharmacophoric approach involving coupling of sulfonamide, amide and amine as three
distinct pharmacophoric entities possessing intriguing structural features such as C=O, S=O, NH2
and NH group. Furthermore we altered the aromatic part with different groups and also used
commercial drugs like amantadine. In addition, we explored the potential of synthesized
compounds against carbonic II anhydrase enzyme and also provided close in silico insights of
binding analysis and chemo-informatics.
2. Experimental
2.1 All chemicals, reagents, were purchased from Sigma-Aldrich Chemical Co. or Merck,
Germany, and were used without further purification. The solvents were dried and distilled prior
5

to use. The Rf values were determined using aluminium pre-coated silica gel plates Kiesel 60F254
from Merck (Darmstadt, Germany). The melting points of the compounds were measured in
open capillaries using a Stuart melting point apparatus (SMP3) and are uncorrected. The FT IR
spectra were recorded on an FTS 3000 MX, Bio-Rad Merlin (Excalibur Model)
spectrophotometer as pure compounds. The 1H and 13C NMR spectra were acquired on a Bruker
NMR spectrometer at 300 MHz and 75.5 MHz using TMS as an internal standard. Mass spectra
were recorded on an Agilent Technologies 6890N gas chromatograph equipped with an inert
mass selective detector (5973 mass spectrometer), and elemental analyses were conducted using
a LECO-183 CHNS analyser.
2.2 Synthesis of 2-chloro-N-(4-sulfamoylphenyl) acetamide (3)
To a stirred solution of 4-aminobenzenesulfonamide (4 mmol) in tetrahydrofuran, at 0-5 °C in ice
jacket, 2-chloroacetyl chloride (4.5 mmol) was added drop wise over a 15-min period. The
reaction mixture was further stirred for half an hour. On completion of reaction, the white
precipitate appeared was filtered, washed and recrystallize with ethanol.
2.3 Synthesis of 2-(substituted phenylamino)-N-(4-sulfamoylphenyl) acetamides (4a-4m)
Suitably substituted aniline (2.016 mmol) were added along with anhydrous potassium carbonate
and a catalytic amount of potassium iodide to the stirred solution of 2-chloro-N-(4-
sulfamoylphenyl) acetamide (3) (0.5g, 2.016 mmol) in the mixture of tetrahydrofuran and
ethanol (10 ml). The reaction mixture was heated under reflux for 6-7 hours. The solid products
thus appeared were filtered, washed and recrystallized with ethanol: chloroform (1:1) mixture.
2-((3-chlorophenyl)amino)-N-(4-sulfamoylphenyl)acetamide (4a):
6

º
1
Light brown solid, m.p.= 261-262 C, yield =64%, Rf = 0.72 (Chloroform: Methanol 4:1); H
NMR (acetoned6): δ (ppm) 9.69 (broad s, 1H, NH), 7.87 (s, 4H, Ar-H), 7.14 (t, 1H, J=8.1 Hz,
Ar-H), 6.71 (t, 1H, J=1.8 Hz, Ar-H), 6.67 (d, 1H, J=8.4, Ar-H), 6.61(d, 1H, J=8.4 Ar-H), 6.53(s,
2H, SO2NH2), 5.80 (t, 1H, NH, J=5.4), 4.0 (d, 2H, CH2, J=5.4). 13C-NMR: (75 MHz Acetone-d6)
δ (ppm) 169.32 (C=O), 149.62, 141.92, 138.79, 134.36, 130.44, 127.07, 119.04, 117.05, 112.43,
111.25, 47.91 (CH2).
N-(4-sulfamoylphenyl)-2-((4-sulfamoylphenyl)amino)acetamide (4b):
º
1
White solid, m.p.= 290-293 C, yield = 61%, Rf = 0.41 (Chloroform: Methanol 4:1); H NMR
(DMSO-d6): δ (ppm) 8.89 (s, 1H, NH), 7.80 (broad s, 8H, Ar-H), 7.53(s, 2H, SO2NH2), 6.65 (s,
2H, SO2NH2) 5.10 (t, 1H, NH, J=5.3), 4.0 (d, 2H, CH2, J=5.3). 13C-NMR: (75 MHz DMSO-d6) δ
(ppm) 172.22 (C=O), 158.72, 142.77, 138.99, 135.66, 133.84, 128.09, 120.03, 116.08, 113.45,
112.35, 49.31 (CH2).
N-(4-sulfamoylphenyl)-2-(p-tolylamino)acetamide (4c):
º
1
Light brown solid, m.p.= 254-256 C, yield = 69%, Rf = 0.63 (Chloroform: Methanol 4:1); H
NMR DMSO-d6): δ (ppm) 9.22 (s, 1H, NH), 7.97 (s, 4H, Ar-H), 7.21 (d, 2H, J=7.9 Hz, Ar-H),
6.50 (d, 2H, J=7.8 Hz, Ar-H), 6.40 (s, 2H, SO2NH2), 5.92 (t, 1H, NH, J=5.2), 4.1 (d, 2H, CH2,
J=5.2,). 13C-NMR: (75 MHz DMSO-d6) δ (ppm) 168.62 (C=O), 144.68, 141.72, 137.59, 128.62,
128.24, 118.09, 113.04, 44.61 (CH2).
2-((4-chlorophenyl)amino)-N-(4-sulfamoylphenyl)acetamide (4d):
º
1
Light brown solid, m.p.= 257-258 C, yield = 71%, Rf = 0.67 (Chloroform: Methanol 4:1); H
NMR (DMSO-d6): δ (ppm) 9.19 (s, 1H, NH), 8.01 (s, 4H, Ar-H), 7.56 (d, 2H, J=7.7 Hz, Ar-H),
6.64 (d, 2H, J=7.7 Hz, Ar-H), 6.53 (s, 2H, SO2NH2), 5.70 (t, 1H, NH, J=5.3), 4.07 (d, 2H, CH2,
7

13
J=5.3). C-NMR: (75 MHz DMSO-d6) δ (ppm) 169.02 (C=O), 146.70, 142.51, 136.39, 131.12,
129.14, 125.81, 117.41, 115.32, 46.71 (CH2).
(S)-2-((2-oxo-2-((4-sulfamoylphenyl)amino)ethyl)amino)pentanedioic acid (4e)
º
1
White solid, m.p.=233-235 C, yield =57%, Rf = 0.46 (Chloroform: Methanol 4:1); H NMR
(DMSO-d6): δ (ppm) 10.6 (broad s, 1H, COOH), 10.4 (broad s, 1H, COOH), 9.33 (broad s, 1H,
NH), 7.81 (d, 2H, J=7.3Hz, Ar-H), 6.97 (d, 2H, J=7.3Hz, Ar-H), 6.63(s, 2H, SO2NH2), 5.70 (t,
1H, NH, J=5.3), 3.91 (d, 2H, CH2, J=5.3). 13C-NMR: (75 MHz Acetone-d6) δ (ppm) 175.12
(C=O), 179.23 (C=O), 167.14, (C=O), 145.52, 138.19, 131.16, 118.04, 64.08(CH), 48.17 (CH2).
31.7,28.32
2-((2-chlorophenyl)amino)-N-(4-sulfamoylphenyl)acetamide (4f):
º
1
Light brown solid, m.p.= 269-271 C, yield = 75%, Rf = 0.75 (Chloroform: Methanol 4:1); H
NMR (DMSO-d6): δ (ppm) 9.49 (s, 1H, NH), 7.91 (s, 4H, Ar-H), 7.59 (d, 1H, J=7.9 Hz, Ar-H),
7.23 (t, 1H, J=7.8 Hz, Ar-H), 6.81 (d, 1H, J=7.8 Hz, Ar-H), 6.41 (t, 1H, J=7.9 Hz, Ar-H), 6.58 (s,
2H, SO2NH2), 4.89 (t, 1H, NH, J=5.1), 4.11 (d, 2H, CH2, J=5.3). 13C-NMR: (75 MHz DMSO-d6)
δ (ppm) 170.01 (C=O), 142.92, 141.67, 135.19, 132.16, 128.21, 126.51, 124.01, 122.29, 117.90,
115.07, 47.53 (CH2).
2-((2,4-dinitrophenyl)amino)-N-(4-sulfamoylphenyl)acetamide (4g):
º
1
Yellow solid, m.p.= 287-288 C, yield = 58%, Rf = 0.43 (Chloroform: Methanol 4:1); H NMR
(DMSO-d6): δ (ppm) 10.09 (s, 1H, NH), 8.91 (s, 1H, Ae-H), 8.63 (d, 1H, J=9.1Hz, Ar-H), 8.01
(s, 4H, Ar-H), 7.43 (d, 1H, J=9.2Hz, Ar-H), ), 6.88 (s, 2H, SO2NH2), 5.41 (t, 1H, J=4.9Hz, NH),
3.89 (d, 2H, J=4.9Hz, CH2); 13C NMR (75 MHz DMSO-d6) δ (ppm) 169.35 (C=O), 146,51,
143.61, 137.25, 136.01, 135.22, 131.06, 129.66, 123.24, 120.45, 119.21, 48.07 (CH2).
8

4-((2-oxo-2-((4-sulfamoylphenyl)amino)ethyl)amino)benzoic acid (4h):
º
1
Dark brown solid, m.p.= 277-278 C, yield = 65%, Rf = 0.61 (Chloroform: Methanol 4:1); H
NMR (DMSO-d6): δ (ppm); 11.53 (broad s, 1H, COOH), 9.84 (s, 1H, NH), 7.93 (m, 4H, Ar-H),
7.78 (d, 1H, J=8.1Hz, Ar-H), ), 6.72 (s, 2H, SO2NH2), 6.61 (d, 1H, J=8.6Hz, Ar-H,) 5.74 (t, 1H,
J=5.2Hz, NH), 4.09 (d, 2H, J=5.2Hz, CH2); 13C NMR (75 MHz DMSO-d6) δ (ppm) 169.46
(C=O), 168.15 (C=O), 149,11, 144.21, 137.25, 131.21, 129.52, 119.16, 118.72, 116.28, 49.17
(CH2).
2-((2,3-dichlorophenyl)amino)-N-(4-sulfamoylphenyl)acetamide (4i):
º
1
Light brown solid, m.p.= 273-274 C, yield = 67%, Rf = 0.52 (Chloroform: Methanol 4:1); H
NMR (DMSO-d6): δ (ppm); 9.74 (s, 1H, NH), 7.82 (d, 2H, J=8.1 Ar-H), 7.68 (d, 2H, J=8.1Hz,
Ar-H), ), 7.24 (d, 1H, J=8.5Hz, Ar-H), 7.11 (t, 1H, J=8.5Hz, Ar-H), 6.43 (s, 2H, SO2NH2), 6.21
13
(d, 1H, J=8.5Hz, Ar-H), 5.02 (t, 1H, J=5.1Hz, NH), 4.34 (d, 2H, J=5.1Hz, CH2); C NMR (75
MHz DMSO-d6) δ (ppm) 169.76 (C=O), 148.67, 145.91, 138.25, 134.45, 133.34, 130.02, 123.16,
121.92, 119.12, 117.28, 49.45 (CH2).
2-((1R,3S,5r,7r)-adamantan-2-ylamino)-N-(4-sulfamoylphenyl)acetamide (4j)
°
1
White solid, m.p.=243-245 C, Yield=58%, Rf = 0.55 62 (Chloroform: Methanol 4:1); H NMR
(DMSO-d6, 300 MHz,); δ (ppm): 9.19 (broad s, 1H, NH), 8.17 (d, 2H, J=7.7Hz, Ar-H), 7.94 (d,
2H, J=7.7 Hz, Ar-H), 6.13(s, 2H, SO2NH2), 5.10 (t, 1H, NH, J=5.2), 3.36 (d, 2H, CH2, J=5.2),
13
2,28 (m, 6H, CH2), 2.18 (m, 3H, CH), 1.56 (m, 6H, CH2) ; C NMR (75 MHz DMSO-d6) δ
(ppm) 167.88 (C=O), 141, 131.88, 129.36, 129.22, (Ar-C), 54.15, 47.23, 40.27, 36.15, 29.12
9

4-((2-oxo-2-((4-sulfamoylphenyl)amino)ethyl)amino)benzenesulfonic acid (4k):
1
White solid, m.p.=279-281 °C, yield,=62%, Rf = 0.39 (Chloroform: Methanol 4:1); H NMR
(DMSO-d6): δ (ppm) 9.92 (s, 1H, NH), 7.91 ( broad s, 8H, Ar-H), 6.93(s, 2H, SO2NH2), 5.20 (t,
1H, J=5.2Hz, NH), 4.03 (d, 2H, J=5.2Hz, CH2 ). 13C-NMR: (75 MHz DMSO-d6) δ (ppm) 171.12
(C=O), 157.22, 146.17, 145.29, 144.66, 135.84, 134.09, 128.13, 125.09, 120.45, 119.15, 49.51
(CH2).
2-((4-nitrophenyl)amino)-N-(4-sulfamoylphenyl)acetamide (4l):
º
1
Light yellow solid, m.p.=284-287 C, yield,=59%, Rf = 0.53 (Chloroform: Methanol 4:1); H
NMR (DMSO-d6): δ (ppm); 9.94 (s, 1H, NH), 8.11 (d, 2H, J=8.9Hz, Ar-H), 7.91 (d, 2H,
J=8.2Hz, Ar-H), 7.72 (d, 2H, J=8.2Hz, Ar-H), 6.73 (d, 2H, J=8.9Hz, Ar-H), 6.56 (s, 2H,
SO2NH2), 5.34 (t, 1H, J=5.6Hz, NH), 4.23 (d, 2H, J=5.6Hz, CH2 ). 13C-NMR: (75 MHz DMSO-
d6) δ (ppm), 171.32, (C=O), 151.24, 141.62, 139.79, 136.06, 135.14, 128.07, 124.04, 119.07,
47.51 (CH2).
2-(naphthalen-1-ylamino)-N-(4-sulfamoylphenyl)acetamide (4m)
º
1
brown solid, m.p.= 271-273 C, yield =69%, Rf = 0.62 (Chloroform: Methanol 4:1); H NMR
(acetoned6): δ (ppm) 9.09 (broad s, 1H, NH), 8.07 (d, 2H, J=6.5Hz, Ar-H), 7.77 (s, 4H, Ar-H),
7.43 (t, 2H, J=6.4 2Hz, Ar-H), 7.01-6.30 (5H, Ar-H), 5.40 (t, 1H, NH, J=5.1), 3.75 (d, 2H, CH2,
J=5.1). 13C-NMR: (75 MHz Acetone-d6) δ (ppm) 167.12 (C=O), 147.62, 143.92, 137.79, 131.36,
129.44, 127.07, 126.04, 125.07, 124.99, 124.70, 118.05, 118.01, 109.43, 46.91 (CH2).
2.3 Carbonic anhydrase assay
10

Carbonic anhydrase inhibition was measured as described previously with some modifications
[23]. 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, 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 buffer and used fresh every time) and 40 µL was added per
well to achieve 0.6 mM concentration per well. Total reaction volume was made to 200 µL.
After30 min incubation at 25 °C contents were 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 independent
experiments. IC50 values were calculated by nonlinear regression using GraphPad Prism 5.0.
Inhibition (%)
Here, the B and S are the absorbance for the blank and samples.
2.4 Free radical scavenging assay
Radical scavenging activity was determined by modifying method by 2, 2-diphenyl-1-
picrylhydrazyl (DPPH) assay [24]. The assay solution consisted of 100 mL of (150 mM) 2,2-
diphenyl-1 picrylhydrazyl (DPPH), 20 µL of increasing concentration of test compounds and the
volume was adjusted to 200 µL in each. This reaction mixture was then incubated for 30 min at
room temperature. Ascorbic acid (Vitamin C) was used as a reference inhibitor. The
measurements were carried out by using a micro plate reader (OPTIMax, tunable) at 517 nm.
11

The reaction rates were compared and the percent inhibition due to the presence of tested
inhibitors was calculated. Each concentration was analyzed in three independent experiments.
3. Methodology
3.1 Repossession of Carbonic anhydrase II from PDB
The three dimensional (3D) crystal structure of carbonic anhydrase II was retrieved form 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 1.10.1 [25]. The stereo-chemical properties, Ramachandran graph and values [26] of
Carbonic anhydrase II structure were assessed by Molprobity server [27], while the
hydrophobicity graph was generated by Discovery Studio 4.1 Client [28]. The protein
architecture and statistical percentage values of helices, beta-sheets, coils and turns were
accessed by using online tool VADAR 1.8 [29].
3.2 In-silico designing of synthesized compounds
The synthesized ligand molecules 4a-4m 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
(https://pubchem.ncbi.nlm.nih.gov/). Moreover, Lipinski’s rule of five was analyzed using
Molsoft and Molinspiraion tools.
3.3 Molecular docking
12

The synthesized ligands 4a-4m were sketched in ACD/ChemSketch tool and minimized by
UCSF Chimera 1.10.1 tool. The molecular docking experiments were performed using PyRx
docking through VINA wizard [30]. The grid box center values were adjusted as for X=10.48,
Y=-55.22 and Z=-26.48), respectively. While, the size parameters values for X=66.60, Y=60.08,
and Z=56.84 were also focused to get the better conformational binding poses. The default
exhaustiveness value was used to maximize the binding conformational analysis. All the
synthesized ligands were docked separately against urease receptor. The Autodock VINA
scoring function equation was employed as mentioned in supplementary data. The predicted
docked complexes were evaluated on the basis of lowest binding energy (Kcal/mol) values and
structure activity relationship (SAR) analyses. The graphical depictions of all the docked
complexes were accomplished by Discovery Studio (2.1.0) and LIGPLOT [31].
4. Results and Discussion
Chemistry
The synthesis of sulfonamide bearing amides and amine functional moieties is illustrated in
Scheme 1. 2-Chloro-N-(4-sulfamoylphenyl)acetamide (3) was obtained by reacting 4-
°
aminobenzenesulfonamide (1) in tetrahydrofuran, with 2-chloroacetyl chloride (2) at 0-5 C.
Suitably substituted aniline in presence of anhydrous potassium carbonate and a catalytic amount
of potassium iodide were treated with (3) in tetrahydrofuran and ethanol to afford the target
molecules (4a-4m) in good yields.
13

14

Scheme 1. Synthesis of 2-(substituted phenylamino)-N-(4-sulfamoylphenyl) acetamide 4a-
4m
The synthesized compounds were characterized through 1H-NMR and 13C-NMR
spectroscopy. The broad and deshielded singlet for amidic N-H was observed around 11-12 ppm.
The singlet at 2-3 ppm was assigned to CH2 in between the amide and amine and the aromatic
13
region appeared at 7-8 ppm. The C-NMR signal for carbonyl of amide appeared in the range
169-160 ppm; those in the range 40-50 ppm value were assigned to CH2, while the aromatic
carbons appeared in the range 120-140 ppm.
Carbonic anhydrase II activity and preliminary structure activity relationship
The results of carbonic anhydrase II (CA-II) has been documented in Table 1. The three 4a, 4d
and 4f derivatives in the series contains chlorine atom tagged with phenyl ring at meta, para and
ortho position respectively and the results of inhibition of CA-II of these three molecules are in
this order; 4a>4d>4f. This reveals that chlorine at meta position is able to polarize the molecule
more effectively compared to para and ortho position. The compounds 4h and 4k possess
carboxylic acid and sulfonic acid respectively and results exhibited that compound 4h showed
better activity compared to 4k. The compound 4g and 4l possess dinitro and mono nitro group
attached at the phenyl ring and compound 4l showed better results compared to 4g and also
compared to other derivatives of the series. However, 4j possess amantadine drug as part of the
molecule and it showed good inhibition but we assumed on the basis of hypothesis (coupling of
two or more pharmacophoric units results in enhanced activity) that compound 4j should exhibit
maximum inhibition but experimental results summarized in Table depict that compound 4l
showed maximum inhibition.
15

Table 1. Carbonic anhydrase II activity (IC50 nM) of compounds (4a-4m)
4a
73.6 ± 1.4
4b
194.3 ± 3.8
214.6 ± 4.2
166.2 ± 3.2
116.9 ± 2.3
182.0 ± 3.6
621.0 ± 12.3
21.9 ± 0.43
75.8 ± 1.5
4c
4d
4e
4f
4g
4h
4i
4j
84.5 ± 1.67
858.0 ± 17.0
17.96 ± 0.36
81.3 ± 1.61
997.1 ± 19.75
4k
4l
4m
Acetazolamide
16

For calculation of IC50 six to eight concentrations were used. IC50 values were calculated by
nonlinear regression using GraphPad Prism 5.0.
Free radical scavenging
All of the synthesized 4a-4m series compounds were evaluated for DPPH free radical scavenging
ability. The compound 4f showed excellent % scavenging potency, other compounds did not
show significant radical scavenging potential even at high concentration (100µg/mL) Figure.1
(DPPH).
Figure.1 Free radical % scavenging activity of synthetic compounds values was represented as
mean ± SEM. All compounds concentrations were 100µg/mL
Chemo-informatic properties and Lipinski Rule (RO5) evaluation of ligands
17

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 1. 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, [32, 33]. The predicted results of compounds showed 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 then 10 and 5 respectively,
[34]. The chemo-informatics analysis justified that all the designed compounds possess <10
HBA and <5 HBD. Moreover, their logP values were also comparable with standard value.
However there are plenty of examples available for RO5 violation amongst the existing drugs
[35, 36]. The predicted chemo-informatics values of all the designed ligand are mentioned in
Table 2.
Table. 2 Chemo-informatics analysis of designed chemical compounds
Mol.
No.
No.
Mol.
PSA
Mol.Vol Drug
Ligands
Wt(g/mol) HBA HBD Logp(mg/L) (A2)
(A3)
Score
0.12
0.76
0.31
1.10
0.29
4a
4b
4c
4d
4e
339.04
384.06
319.10
339.04
359.08
4
7
4
4
9
4
6
4
4
6
2.08
0.19
1.77
2.08
-1.79
84.23 279.51
134.83 309.23
84.23 285.52
84.23 279.44
142.24 309.58
18

4f
339.04
395.05
349.07
373.01
363.16
385.04
350.07
355.10
4
8
6
4
5
7
6
4
4
4
5
4
4
5
4
4
1.96
0.70
1.08
2.56
1.81
-0.47
1.10
2.70
83.53 278.32
159.75 316.79
112.63 297.68
83.53 293.99
85.89 346.17
126.64 305.15
122.49 290.21
83.26 314.81
0.33
-1.18
1.24
0.61
1.17
0.65
-0.18
0.03
4g
4h
4i
4j
4k
4l
4m
Abbreviation: HBA= No of hydrogen bond acceptor, HBD= No of hydrogen bond donor, LogP=
lipophilicity of partition coefficient, LogS= lipophilicity of water, PSA= polar surface area,
MR=Molar refractivity, PZ=polarizability
Molecular docking and binding energy analysis
The docked complexes of all the compounds 4a-4m against carbonic anhydrase II were analyzed
separately and evaluated on the basis of minimum energy values and ligand interactions pattern.
Results showed that all compounds 4a-4m showed good binding energy value and exhibited in
the active region of 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 ligands possess good efficient energy
values and have no big energy fluctuations difference. The comparative docking analysis and
19

inhibition constant (IC50) value 0.01796±0.00036M justified that 4l has good therapeutic
potential as compared to all other compounds.
Table 3. Docking results of synthesized compounds
Binding
Docking complexes
Affinity
-6.7
-7.5
-6.7
-6.6
-7.6
-6.8
-6.9
-7.1
-6.9
-6.8
-7.2
-6.7
-7.6
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
20

Binding analyses of synthesized compounds against carbonic anhydrase II
The ligands-protein binding analyses showed that 4l confined in the active binding pocket of
target protein as mentioned in Figure 2. The CA II has an active site cleft (15 Å in diameter and
15 Å deep), and contains a Zinc ion that is coordinated in a tetrahedral geometry with three
histidine residues (His94, His96 and His119) and a water molecule/hydroxide ion. The 4l
receptor docked complex reveals the good conformational state with hydrogen bond interactions
within the receptor binding pocket. The docking result of 4l receptor docked complex showed
that three hydrogen bonds were observed at His63, Asn66 and Thr197 residues, respectively. The
amino carbonyl moiety of functional group in A12 interacts with His63 with bond distance
2.49Å while other form another hydrogen bond against Asn66 with bond length 1.92Å,
respectively. Similarly, another hydrogen bond was observed between amino group of 4l and
Thr197 having bond length 2.36 Å. Single hydrophobic interaction was also seen between His93
and oxygen moiety of 4l with bond length 3.06 Å. No π-π stacking interactions were observed
between ring structure of ligands and aromatic residues of target protein.
Figure 2. Docking interaction 4l with receptor molecule. A) The protein structure is represented
in grey color in surface format to show the binding pocket of target protein. B) Docking complex
21

A12 is shown in grey ribbon with interior brown color, while the interacted residues are justified
in light dark khaki color. C) The closer view of binding interaction. The ligand molecule is
depicted in yellow color while their functional groups such as oxygen, nitrogen and sulphur are
showed in red, blue and yellow colors respectively. Amino acids are highlighted in dark khaki
color and red and purple dotted lines justifies the hydrogen and hydrophobic bindings with
distance mentioned in angstrom (Å). Zinc metal is represented in grey circle.
5. Conclusions
To test the hypothesis that coupling of two more pharmacophoric units results in the
enhancement of biological activity, we envisioned coupling of sulfonamide, an amide and amine
can result in the higher biological activity compared to the already known commercial drugs. In
this connection, a new series of compounds 4a-4m was synthesized and characterized through
spectroscopic techniques and subjected to carbonic anhydrase II inhibition. Among the series
compound 4l exhibited highest activity (0.01796±0.00036M) compared to the other memebers
of series as well as the standard acetazolamide. The Lipinski’s rule and chemo-informatics
evaluation revealed that molecules possess significant hydrogen bonding sites which can enable
them to bind in the active site of target protein. The binding analysis of ligand-protein that
compound 4l possess minimum binding energy (-6.7 KJ/mol) and shows hydrogen bonding
interaction with His63, Asn66 and Thr197 amino acid residues. To sum up, present investigation
provides an incentive to further explore the structure activity relationship and design safe and effective
inhibitors of carbonic anhydrase II enzyme.
Conflict of interest
Authors declare no any conflict of interest
22

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Highlights
 Hybrid pharmacophore containing sulfonamide, amide and amine designed and
synthesized.
 All the derivatives 4a-4m showed better inhibition than acetazolamide, with 4l exhibiting
momentous inhibition
 The chemo-informatics and ligands-protein binding analyses carried out.
28