Sulfonamide-Linked ciprofloxacin, sulfadiazine and amantadine derivatives as a novel class of inhibitors of jack bean urease; synthesis, kinetic mechanism and molecular docking

Article abstract of DOI:10.3390/molecules22081352

Sulfonamide derivatives serve as an important building blocks in the drug design discovery and development (4D) process. Ciprofloxacin-, sulfadiazine- and amantadine-based sulfonamides were synthesized as potent inhibitors of jack bean urease and free rad

Full text of DOI:10.3390/molecules22081352

molecules
Article
Sulfonamide-Linked Ciprofloxacin, Sulfadiazine and
Amantadine Derivatives as a Novel Class of
Inhibitors of Jack Bean Urease; Synthesis,
Kinetic Mechanism and Molecular Docking
Pervaiz Ali Channar 1, Aamer Saeed 1,
*
ID , Fernando Albericio 2,3 ID , Fayaz Ali Larik 1,
*
,
ID
Qamar Abbas 4,5, Mubashir Hassan 4, Hussain Raza 4 and Sung-Yum Seo 4
1
Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan; mrpervaiz@gmail.com
Department of Organic Chemistry, University of Barcelona, 08028 Barcelona, Spain; albericio@ukzn.ac.za
CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine,
Barcelona Science Park, University of Barcelona, 08028 Barcelona, Spain
Department of Biological Sciences, College of Natural Sciences, Kongju National University,
56 Gongjudehak-Ro, Gongju, Chungnam 314-701, Korea; qamar.abbas.qau@gmail.com (Q.A.);
mubashirhassan_gcul@yahoo.com (M.H.); hussain_solangi@yahoo.com (H.R.);
dnalove@kongju.ac.kr (S.-Y.S.)
2
3
4
5
Department of Physiology, University of Sindh, Jamshoro 76080, Pakistan
Correspondence: aamersaeed@yahoo.com (A.S.); fayazali@chem.qau.edu.pk (F.A.L.);
Tel.: +92-519-064-2128 (A.S.); Fax: +92-519-064-2241 (A.S.)
*
Received: 5 July 2017; Accepted: 9 August 2017; Published: 16 August 2017
Abstract: Sulfonamide derivatives serve as an important building blocks in the drug design discovery
and development (4D) process. Ciprofloxacin-, sulfadiazine- and amantadine-based sulfonamides
were synthesized as potent inhibitors of jack bean urease and free radical scavengers. Molecular
diversity was explored and electronic factors were also examined. All 24 synthesized compounds
exhibited excellent potential against urease enzyme. Compound 3e (IC50 = 0.081
6a (IC50 = 0.0022 ± 0.0002 µM), 9e (IC50 = 0.0250 0.0007 M) and 12d (IC50 = 0.0266
were found to be the lead compounds compared to standard (thiourea, IC50 = 17.814
±
0.003
0.0021
0.096
µ
M),
M)
M).
±
µ
±
µ
±
µ
Molecular docking studies were performed to delineate the binding affinity of the molecules and
a kinetic mechanism of enzyme inhibition was propounded. Compounds 3e 6a and 12d exhibited
,
a mixed type of inhibition, while derivative 9e revealed a non-competitive mode of inhibition.
Compounds 12a, 12b, 12d, 12e and 12f showed excellent radical scavenging potency in comparison
to the reference drug vitamin C.
Keywords: jack bean urease inhibition; kinetic mechanism; molecular docking; sulfonamides;
drug derivatives; drug discovery
1. Introduction
Ureases (EC 3.5.1.5; urea amidohydrolase) belong to a super group of amidohydrolases and
phosphotriestreases, heteropolymeric enzymes with the dynamic site containing two nickel (II) atoms,
found in extensive amounts in nature among plants, microbes, organisms, green growth and spineless
creatures [1–3]. Urease-delivering microscopic organisms have harmful effects on human health.
Thus, in human beings the ureas of Helicobacter pylori cause afflictions of the gastrointestinal and
urinary tract, for example, stomach disease and peptic ulcers [4,5].
Ciurli et al. proposed a productive and workable enzymatic mechanism [6,7]. The dynamic focus
of urease is relied on trapping three water molecules and a hydroxide ion connects between two nickel
Molecules 2017, 22, 1352; doi:10.3390/molecules22081352
www.mdpi.com/journal/molecules

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atoms [
The loosely bound urea molecule collapses in a tetrahedral fashion with the release of the carbamate
group which eventually cleaves into an ammonia molecule [ ]. The release of excess ammonia
8]. Urea possesses two binding sites and is capable of forming hydrogen bonding linkages.
9
furnishes the suitable conditions for the survival of H. pylori in the stomach [10]. H. pylori causes
several stomach-related disorders such as urolithiasis, pyelonephritis, hepatic encephalopathy, hepatic
unconsciousness and urinary catheter encrustation [11]. The therapeutic treatment of Helicobacter
pylori has been summarized in a review by Boer et al. [12]. Ureases have a long storied history and
research on the toxicity and multifunctionality of ureases is work in progress. Carlini et al. have
comprehensively reviewed the mechanism and function of ureases [13]. Urease inhibitors play a
pivotal part in the inhibition of the harmful effects of urease enzyme and substantially improve
human health [14]. Moreover, urease inhibitors assist in the design of drugs against stomach
ulcer disorders [15,16]. Urease has assorted capacities and its inhibition has received exceptional
consideration in the course of recent years and numerous urease inhibitors have been described.
Among these are hydroxamic corrosive subordinates [17], hydroxyurea [18], hydroxamic acids [19],
phosphorodiamidates, imidazoles, for example, rabeprazole, lansoprazole, omeprazole, quinines,
thiol derivatives, and phenols, Schiff base and thiourea derivatives [20]. Sulfonamides constitute an
important class of organic compounds that possess a broad spectrum of biological activities such as
antibacterial, high-ceiling diuretic, hypoglycemic, antithyroid, anti-inflammatory and antiglaucoma
effects [21–27]. Noreen et al. recently reported thiophene-tagged sulfonamides as µg/mL concentration
urease inhibitors. Mojzych et al. published pyrazoletriazine- based sulfonamides as dual potent
inhibitors of urease and tyrosinase and their synthesized derivatives showed better potential than
standard thiourea, with IC50 values in the micromolar range [28–30].
Sulfonamides can easily be synthesized by the reaction of sulfonyl chlorides with amines in a
basic medium. However, a number of different methods for the synthesis of sulfonamides have been
described in the literature. The straightforward synthetic routes and extended applications in the
pharmaceutical and biological field provide incentive to explore and design the role of commercial
drugs based sulfonamides as urease inhibitors.
Herein, the exploration of novel sulfonamides based drug derivatives, as significant inhibitors of
jack bean urease, are described. We thus extended the range of commercial drugs like ciprofloxacin,
sulfadiazine, amantadine and thiosemicarbazide (Figure 1).
Figure 1. Structures of some synthetic potential urease inhibitor molecules.
The potential of commercial drugs as inhibitors of urease has not been explored in enzymology.
All three drugs mentioned in Figure 1 are different from one another. However, these drugs
contains intriguing structural features which can show strong binding affinity with the target protein.
These drugs share a common nucleophilic behavior owing to the presence of electron rich nitrogen
atoms. Prior to the current research account, the scope of these drugs has not been extended to
urease inhibition. It was hypothesized that variation or structural modification in these commercial
drugs could lead to the development of efficient and side effect-free potent inhibitors of urease.
In order to test this hypothesis we envisioned uncovering the potential of some marketed drugs.
Moreover, the synthetic molecule thiosemicarbazide was also examined to evaluate the role of
small organic molecules as inhibitors of urease. Commercial drugs used in this research work are

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endowed with complex structural groups which could lead to strong binding in the active site of the
target protein.
The sulfonamide derivatives have been considered as suitable candidates for the carbonic
anhydrase inhibition assay. We took a step further to explore the role as urease inhibitors of
sulfonamides, a privileged class of organic compounds.
2. Results
The synthetic routes to compounds 3a–3f/6a–6f/9a–9f/12a–12f are shown in Scheme 1. The new
series of sulfonamide-based drugs and thiosemicarbazide-based sulfonamides were synthesized in a
single step using sulfonyl chloride as an electrophilic reagent. The amine group-bearing versatile drugs
ciprofloxacin, amantadine, sulfadiazine and thiosemicarbazide were reacted with suitably substituted
sulfonyl chlorides to construct sulfonamide linkages. For sulfadiazine where the amine function is less
reactive, reflux conditions in pyridine were required. For the rest, just room temperature. In all cases,
the reaction proceeded smoothly with excellent yields (>70%).
Scheme 1. Synthesis of ciprofloxacin-appended sulfonamide derivatives 3a
drug derivatives 6a 6f, amantadine-linked sulfonamides 9a 9f and thiosemicarbazide sulfonamide
derivatives 12a–12f.
–3f, sulfadiazine
–
–
2.1. Jack Bean Urease Inhibition Assay and Structure Activity Relationship (SAR)
Novel sulfonamide based drug-derivatives were screened in a jack bean urease enzyme inhibition
assay. The structural resemblance of the synthesized derivatives with urease enzyme resulted in
promising activity. Thiourea was taken as reference drug (Table 1). The synthesized derivatives
showed different patterns of chemical reactivity, depending upon the nature of the groups attached
with the phenyl ring. The urease inhibition results revealed that these compounds display significant
inhibition at low dosis. Among the tested drug derivatives, the ciprofloxacin derivative 3e, sulfadiazine
derivative 6a, amantadine derivative 9e and thiosemicarbazide derivative 12d were found to be the
best, with IC50 values of 0.0453
respectively. The drug structure played a pivotal part in the urease enzyme inhibition assay.
±
0.0016, 0.00223
±
0.00021, 0.0250
±
0.00073 and 0.0266 ± 0.0021 µM
,

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Generally, ciprofloxacin-tagged sulfonamides showed better potency compared to sulfadiazine and
amantadine derivatives.
Table 1. The inhibitory effect of sulfonamide based drug series on urease (jack bean) activity.
Urease Activity
Urease Activity
Compound
Compound
IC50 ± SEM (µM)
IC50 ± SEM (µM)
3a
3b
3c
3d
3e
3f
6a
6b
6c
0.081 ± 0.003
0.078 ± 0.003
0.110 ± 0.004
0.082 ± 0.003
0.045 ± 0.001
0.073 ± 0.004
0.002 ± 0.000
0.233 ± 0.011
0.141 ± 0.007
0.095 ± 0.004
0.174 ± 0.009
0.127 ± 0.0076
17.814 ± 0.096
9a
9b
9c
9d
9e
0.2888 ± 0.015
0.02757 ± 0.001
0.01776 ± 0.000
0.0533 ± 0.001
0.0250 ± 0.000
0.0558 ± 0.001
0.2254 ± 0.006
0.141 ± 0.004
0.186 ± 0.007
0.026 ± 0.002
0.096 ± 0.0040
0.067 ± 0.0029
9f
12a
12b
12c
12d
12e
12f
6d
6e
6f
Thiourea
SME (standard error of the mean).
The nature of the group associated with the phenyl ring greatly influenced the biological activity
results. Compound 3e which bears two methyl substituents at the meta position is more potent
compared to 3a where they are located at the para position. Halogens differ in inhibition results,
possibly due to inductive effects, and interestingly a chlorine atom linked at the phenyl ring ortho
position exhibited better activity. Electron releasing groups showed moderate activity.
2.2. Kinetic Mechanism
Presently four compounds 3e, 6a, 9e and 12d (the most potent from each group) were studied
for their mode of inhibition against urease. The capability of these inhibitors to restrain the
enzyme-substrate complex and free enzyme was resolved as far as enzyme inhibitor (EI) and enzyme
substrate complex (ESI) constants individually. The active investigations of the catalyst by the double
reciprocal plot of 1/Velocity versus 1/[Substrate] within the sight of various inhibitors doses gave a
progression of straight lines as they appear in Figure 2. The outcomes of inhibitor 9e provide a sequence
of conventional positions, all of which crossed at the identical point on the x-axis Figure 2, (A). The
analysis showed that Vmax decreased to another dose while that of Km continues as before accordingly
to the increment in the doses of compound 9e. This conduct showed that compound 9e represses
urease non-competitively to frame enzyme inhibitor (EI) complex. An optional plot of slope against
centralization of 9e indicated a consistent EI separation (Ki) Figure 2, (B). The results of Figure 2, (A)
showed that compounds 3e
that Vmax decreased with increasing Km in the presence of increasing concentrations of compounds
3e 6a and 12d, respectively. This mode of action of inhibitors 3e 6a and 12d pointed out that they
, 6a and 12d intersected within the second quadrant. The analysis showed
,
,
stops the enzyme by a dual method: first competitively by forming a urease inhibitor complex and
second by interfering with the urease-substrate (urea)-compound composite, known as noncompetitive
mode. The second graph between inhibitor 3e
dissociation constant constants Ki Figure 2, (B). Whereas enzyme substrate inhibitor dissociation
constants Ki0 were presented by another graph plotting doses of inhibitors 3e
6a and 12d versus
intercept Figure 2, (C). A lesser value of Ki than Ki0 indicated the solid attachment of enzyme and
inhibitors 3e 6a and 12d that indicates more competitive than noncompetitive behavior (Table 2). The
kinetic constant and inhibition constant data are listed in Table 2.
, 6a and 12d versus slope displayed enzyme inhibitor
,
,

Molecules 2017, 22, 1352
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Figure 2. Double reciprocal plot for inhibition of urease in the existence of inhibitor 3e: (
were 100, 50, 25, 12.5, 6.25 and 3.125 mM; ( ) The graph indicates the plot of the slope ( ) the vertical intercepts versus inhibitor 3e concentrations to determine inhibition
constants. Double reciprocal plot plots for inhibition of urease in the presence of Compound 6a: ( ) Concentrations of 6a were 0.00, 0.0011, 0.0022 and 0.0044 M.
Substrate urea concentrations were 100, 50, 25, 12.5, 6.25, and 3.125 mM; ( ) The insets represent the plot of the slope ( ) the vertical intercepts versus inhibitor 6a
concentrations to determine inhibition constants. Lineweaver–Burk plots for inhibition of urease in the presence of Compound 9e: ( ) Concentrations of 9e were 0.00,
0.015, 0.03 and 0.06 M. Substrate urea concentrations were 100, 50, 25, 12.5, 6.25, and 3.125 mM; ( ) The insets represent the plot of the slope. Lineweaver–Burk plots for
inhibition of urease in the presence of Compound 12d: ( ) Concentrations of 12d were 0.013, 0.026, 0.052, 0.104 and 0.208 M. Substrate urea concentrations were 100, 50,
25, 12.5, 6.25 and 3.125 mM; ( ) The insets represent the plot of the slope ( ) the vertical intercepts versus inhibitor 12d concentrations to determine inhibition constants.
A) Doses of 3e were 0.025, 0.05, 0.01 and 0.02 µM. Substrate urea concentrations
B
C
A
µ
B
C
A
µ
B
A
µ
B
C

Molecules 2017, 22, 1352
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Table 2. Kinetic parameters of the jack bean urease for urea activity in the presence of different
concentrations of 3e, 6a, 9e and 12d.
0
Code
Dose (µM)
Vmax (∆A/Min)
Km (mM)
Inhibition Type
Ki (µM)
Ki (µM)
0.025
0.05
0.01
0.02
0
0.00337
0.00273
0.00233
0.001632
0.0204
0.606
1.923
3.389
5.263
5
0.016
0.195
0.0016
—
Mixed-inhibition
3e
0.0011
0.0022
0.0044
0
0.00769
0.00493
0.00351
0.0114
6.666
6.896
7.518
4.166
0.0011
0.0225
0.053
Mixed-inhibition
Noncompetitive
6a
9e
0.015
0.03
0.06
0.00127
0.00108
0.00085
0.00299
4.166
4.166
4.166
1.754
0.013
0.026
0.052
0.104
0.208
0.00213
0.00194
0.0016
0.001
2.439
2.857
3.174
3.773
0.125
Mixed-inhibition
12d
Vmax = the reaction velocity; Km = Michaelis–Menten constant; Ki =EI dissociation constant; Ki0 = ESI dissociation
constant; — = Not calculated.
3. Discussion
Drug discovery is an intriguing process, which cannot be attempted/faced using just a single
strategy. Several groups and important pharmaceutical industries have focused their efforts in the
“repurposing” strategy which is basically based on discovering new uses for known drugs [31].
In our current medicinal chemistry programs, we have gone a step further. Thus, we propose the
manipulation of known drugs. As proof of concept, we have chosen the sulfonamidation reaction,
because sulfonamides are present in many known drugs [32]. As drugs, we have chosen three which
contain free amino functions—ciprofloxacin, sulfadiazine and amantadine—and thiosemicarbazide,
which although simple, is a motif also present in several drugs [33,34].
3.1. Structural Evaluation of the Target Protein
Jack bean urease consists of four domains (A, B, C and D) having different amino acid lengths.
Two nickel atoms are also present in the active binding region of the targeted protein. The overall
Volume Area Dihedral Angle Reporter (VADAR) analysis showed that the urease architecture contains
with 27% helices, 31%
of residues were present in favored regions which shows the precision and accuracy of phi (
) angles among the coordinates of jack bean urease. The Ramachandran and hydrophobicity graphs
β
sheets and 41% coils. The predicted Ramachandran plots indicated that 97.5%
φ) and psi
(ψ
are presented in the Supplementary Data (Figures S1 and S2).
3.2. Biochemical Properties and Rule of Five (RO5) Validation of Synthesized Compounds
The basic biochemical properties of all compounds 3a–f, 6a–f, 9a–f and 12a–f were predicted
using computational tools. Table 3 showed the predicted properties like molecular weight (g/mol),
molar refractivity (cm3), density (g/cm3), polarizability (cm3) and polar surface area (PSA) (Å2) values
of all ligands. The molar refractivity and molecular lipophilicity properties of drug molecules are
significant in receptor binding, bioavailability and cellular uptake. It has been shown that PSA is very
helpful parameter for drug absorption prediction in drug discovery [35]. The literature study reported
the standard values for molar refractivity and molecular weight (40 to 130 cm3) and (160 to 480 g/mol),
respectively [36
,37]. The comparative analyses revealed that the predicted values of all the synthesized
compounds were comparable with standard values.

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Table 3. Physical properties of the synthesized compounds.
Properties
Mol. weight (g/mol)
No. HBA
3a
485
6
3b
517
8
3c
547
6
3d
539
6
3e
499
6
3f
531
8
6a
404
6
6b
436
8
6c
466
6
6d
466
6
6e
457
6
6f
418
6
9a
305
3
9b
337
5
9c
367
3
9d
359
3
9e
319
3
9f
351
5
12a
245
4
12b
277
6
12c
307
4
12d
298
4
12e
259
4
12f
291
6
No. HBD
1
2
1
1
1
2
2
3
2
2
2
2
1
2
1
1
1
2
4
5
4
4
4
5
Mol. LogP
Mol. PSA (A2)
3.5
2.7
4.9
78
0
4.3
78
0
3.9
78
0
2.9
116
0
2.3
102
0
1.4
140
0
3.7
101
0
3.7
101
0
3.1
102
0
2.7
102
0
4.3
41
0
3.5
80
0
5.7
41
0
5.1
41
0
4.7
41
0
3.8
79
0
1.1
73
0
0.29
111
0
2.5
72
0
1.8
73
0
1.50
73
0.5
111
0
78.3 116
Stereo centers
0
0
0
Mol. Vol (A3)
470
122
69
479
124
79
525
142
67
1.4
56
482
127
73
1.6
50
491
127
67
1.4
50
500
128
76
1.5
51
327
101
74
335
102
87
382
121
71
382
121
71
336
106
79
348
105
71
312
83
51
1.2
33
321
85
62
1.4
33
363
104
57
1.2
41
321
88
58
1.4
35
333
88
50
1.2
35
342
90
60
1.3
35
202
62
65
1.4
24
212
64
258
82
63
1.3
32
0.12
5
211
67
73
1.6
26
224
67
232
68
77
1.5
27
Molar Refractivity (cm3)
Surface tension (dyne/cm)
Density (g/cm3)
Polarizability (cm3)
Drug Score
84
61
1.4
48
1.6
49
1.4
40
1.6
40
1.4
48
1.3
46
1.6
42
1.4
42
1.6
25
1.3
26
0.92 0.59 1.07 0.80 1.16 0.46
−
0.14
6
−
0.64
7
0.12
7
0.13
7
0.04
6
0.21
6
−
0.85
3
−
0.61
3
−
0.54
4
−
0.82
3
−
0.30
3
−
1.00
4
−
0.06
4
−
0.01
5
−
0.39
4
0.23
4
−
0.70
4
No. Rotatable bonds
Lipinski Rule
5
6
6
5
5
6
Yes
No
No
No
Yes
No
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
Yes
HBA: hydrogen bond acceptors; HBD: hydrogen bond donors; PSA: polar surface area.

Molecules 2017, 22, 1352
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Moreover, irrespective of their higher molecular weight (g/mol) and molar refractivity (cm3)
values, they showed very good drug-likeness scores. Any single non-ring bond, bonded to a
nonterminal heavy (i.e., non-hydrogen) atom is called rotatable bond. The calculated number of
rotatable bonds showed molecular flexibility. It has been observed that rotatable bond number is very
good descriptor for oral bioavailability drugs [37].
Furthermore, all the compounds were validated by Lipinski’s rules. It has been observed that orally
active drugs typically contain no more than 10 hydrogen bond acceptors (HBA) and five hydrogen bond
donors (HBD). Moreover, the logP and molecular mass value should be less than 5 and 500 g/mol,
respectively. It has been shown that the compounds with HBA and HBD results exceeding these values
display poor permeation [38]. Hydrogen-bonding capacity is considered a significant parameter for drug
permeability. Our results indicated that the all synthesized compounds possess <10 HBA, <5 HBD and
<5 logp values which were comparable with standard values. However, only few compounds possess
a molecular weight value >500 g/mol which does not follow the RO5. The reported study showed
that molecules with poor absorption are more likely to be observed upon Lipinski violation. However,
multiple examples of RO5 violations are known amongst existing drugs [39,40].
3.3. Binding Energy Evaluation of Synthesized Compounds
To predict the best fitted conformational position of the synthesized compounds 3a–f, 6a–f, 6a–f
and 12a–f against the targeted protein, their generated docked complexes were analyzed on the basis
of minimum energy values (Kcal/mol) and bonding interaction patterns (hydrogen/hydrophobic).
Among the four different ligand structures, the docking results justified the fact that compounds
3a–f and 6a–f were the most active with better binding energy values as compared to 9a–f and 12a–f.
The comparative analysis showed that 6b has the highest energy value (9.0 Kcal/mol) complex.
Compound 6a also has a good binding energy affinity (8.6 Kcal/mol). The docking results showed
that compounds 6a
compounds like 3b
7.2 and 7.5 Kcal/mol, respectively). However, all the other compounds also exhibited good binding
–
f
have more binding potential against the target protein. Similarly, some other
,
9c 9f 12c and 12d also possess good binding energy values ( 8.9, 8.5, 8.5,
,
,
−
−
−
−
−
energy values (Kcal/mol) against the receptor molecule. A literature study showed that the standard
error mean of docking energy for Autodock is 2.5 kcal/mol. Therefore, among all compounds the
energy value difference was no more than 2.5 Kcal/mol. However, the selection of compounds was
also confirmed on the basis of in vitro analysis to categorize the compounds and further perform
a dynamic simulation study. Our computational analysis also showed a good association with the
in-vitro study and a graphical depiction is provided in Figure 3.
Figure 3. The graphical depiction of docking energy values.

Molecules 2017, 22, 1352
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3.4. Urease Binding Pocket and Ligand Pose Analysis
The docked complexes were further analyzed on the basis of hydrogen and hydrophobic binding
interactions. Docking analysis showed that the four best compounds were confined in the active
binding region of the receptor molecule with different conformational poses. The four best docked
compounds’ (3e, 6a, 9c and 12d) poses in the binding region of urease with their interaction bonding
patterns are shown in (Figure 4). All the other docking complexes are depicted in the Supplementary
Data (Figures S3–S22).
Figure 4. Binding pocket of urease with binding position of the four best synthesized compounds are
illustrated in the center. The individual docking results of the best four compounds are labelled in the
surrounding circles. In all docking complexes the urease protein is labelled in gray color, with interior
color olive drab while the interacting residues are mentioned in purple color. The most active and
potent compound 6a in the docking complex is shown in khaki color, while the other compounds
(3e, 9e and 12d) are represented in salmon, coral and sea green, respectively. The sulfur group in all
compounds is highlighted in yellow, while the oxygen and amino groups are represented in red and
blue, respectively.
3.5. Structure Activity Relationship and Interacting Residues
The hydrogen and hydrophobic interactions are very important in drug protein docking.
Prior research data showed that hydrogen bonds with donor-acceptor distances of 2.2–2.5 Å are
considered as strong bonds, while, bond distances from 2.5 to 3.2 Å are considered moderate, and bond

Molecules 2017, 22, 1352
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lengths ranging from 3.2–4.0 Å are considered as weak interactions [40]. In detail, the structure activity
relationship (SAR) study showed that in 6a docking two hydrogen bonds were observed against
His593, with bond distances of 2.30, 2.60 Å, respectively [41]. However, another showed that the
default parameter for the distance between the hydrogen bond donor and the acceptor was 3.2 Å [42].
On average, a hydrogen bond with a distance >3.5 Å is considered as weak and a hydrogen bond
with a distance <3.5 Å is considered strong [43]. A single hydrophobic interaction was also seen with
Ala636, showing a bond length of 4.03 Å. The amino group of the benzene ring and oxygen moiety of
the sulfur interact directly with His593 by a hydrogen bond interaction. Similarly, the benzyl methyl
group forms a hydrophobic interaction within the active binding region of the target protein. In the 3e
docking complex three hydrogen bonds were observed. The oxygen and methoxy functional groups
of 3e form hydrogen bonds with Arg439 and Ala636 with bonding distances of 3.06, 2.92 and 4.40 Å,
respectively. Compounds 9e and 12d are also involved in hydrogen bonding interactions with target
protein residues. The oxygen moiety of 9e forms double hydrogen bonds with Arg439 and His594 with
bonding distances of 2.08 and 2.72 Å. Similarly, in 12d docking a single hydrogen bond was observed
between Arg439 and an oxygen molecule of the ligand with a bonding distance 1.78 Å. It has been
observed that Arg439 was most common interacting residues in all docking results. Literature data also
verified the importance of these residues in bonding with other urease inhibitors which strengthens our
docking results. No ligand interaction was observed with the Ni2+ due to the bulky ligand structures.
However, the interaction patterns of our docking results were correlated with published results which
strengthens the significance of our docking experiments [44–48]. The comparative binding energy and
SAR analysis showed the significance of 6a that may be considered as a potent inhibitor targeting jack
bean urease.
3.6. Radius of Gyration and Protein Stability
The radius of gyration (Rg) prediction was also carefully observed to analyze the compactness
of the target protein. The generated graph results indicated that the Rg value is very stable at
around 1.83 nm throughout the simulation time (0–10,000 ps). The comparative analysis showed that
the 6a graph line was the most stable and showed little fluctuations compared to other complexes.
The Rg time graph showed that the residual backbone and the folding of the receptor protein was
steadily stable after binding with the inhibitors (Figure 5). The solvent accessible area (SASA)
and χ2 distribution/dihedral order of residual fluctuations of all complexes are presented in the
Supplementary Data (Figures S23 and S24).
Figure 5. Radius of gyration (Rg) graphs of 3e
, 6a, 9e and 12d docked complexes are indicated in
purple, red, green and blue colors, respectively, on a 0–10,000 ps time scale.

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4. Materials and Methods
4.1. Chemicals and Instruments
All the chemicals and reagents were purchased commercially and used without further
purification. All the solvents used were dried and distilled prior to use. Melting points were
recorded using a digital Gallenkamp model MPD.BM 3.5 apparatus (SANYO, Tokyo, Japan) and
are uncorrected. 1H- and 13C-NMR spectra were recorded in DMSO-d6 at 300 MHz and 75 MHz,
respectively, using a Bruker (Rheinstetten, Germany) spectrophotometer. IR spectra were recorded on
an IR 460 spectrophotometer (Shimadzu, Rheinstetten, Germany) as KBr pellets. Elemental analyses
were conducted using a LECO-183 CHNS analyzer (Rheinstetten, Germany). Bioactivities were
determined at the Department of Biological Sciences, College of Natural Sciences, Kongju National
University. Thin layer chromatography (TLC) was conducted on 0.25 mm silica gel plates (60 F254,
Merck, Rheinstetten, Germany). Visualization was made with ultraviolet light. Reagents were obtained
commercially and used as received.
4.2. Synthetic Procedures
New and modern synthetic protocols were used for the synthesis of sulfonamide derivatives [49].
Sulfonyl chloride (0.5 g, 1.85 mmol) in chloroform (10 mL) was added dropwise to a solution of
ciprofloxacin/amantadine/sulfadiazine or thiosemicarbazide (0.28 g, 1.85 mmol not valid for all
these substrates) and pyridine (0.77 mL) in chloroform (5 mL) and stirred for 48 h. The reaction
mixture was washed successively with 10% citric acid (5–10 mL), 10% NaHCO3 (5–10 mL) and water
(3–10 mL), dried with MgSO4 and concentrated in vacuo. Purification by flash column chromatography
(30% hexane–ethyl acetate) yielded the crude compound as a yellow oil. The oil was further purified
by recrystallization from chloroform and a few drops of n-hexane. The characterization data for the
compounds is given below.
4.3. Characterization Data
1-Cyclopropyl-6-fluoro-7-(4-((3-nitrophenyl)sulfonyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3a). White solid; yield: 76%; m.p.: 225–227 ◦C; Rf: 0.63 (1:1 petroleum ether–ethyl acetate); FTIR
(neat, cm−1): 3135 (Csp2-H), 1663 (C=O), 1589, 1541 (Ar C=C), 1487 (N=O), 1251 (C=S); 1H-NMR:
δ
(ppm) 15.09 (s, 1H, COOH), 8.80 (s, 1H, ArH), 8.39 (d, 1H, ArH, J = 8.8 Hz), 8.29 (s, 1H, Ar-H), 8.21
(d, 1H, Ar-H, J = 8.6 Hz), 7.93 (s, 1H, Ar-H), 7.71–7.67 (m, 1H, ArH), 7.51 (s, 1H, C=CH), 3.69–3.56
(m, 1H, CH), 3.19–3.15 (m, 4H, CH2), 2.64–2.59 (m, 4H, CH2), 1.11 (d, 4H, CH2, J = 6.8 Hz); 13C-NMR:
δ
(ppm) 199.0 (ketone C=O), 173.0 (acid C=O), 162.95, 151.1, 149.8, 146.7, 139.6, 134.5, 133.1, 132.6, 130.3,
129.4, 127.0, 114.6, 113.5, 51.7, 42.0, 34.6, 11.7. Anal. Calcd. for C24H24FN3O5S: C, 59.39; H, 4.96; N,
8.69; S, 6.62 found: C, 59.12; H, 4.84; N, 8.24; S, 6.48.
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-tosylpiperazin-1-yl)-1,4-dihydroquinoline-3-carboxylic acid (3b). White
solid; yield: 80%; m.p.: 160–162 ◦C; Rf: 0.61 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1):
3030 (Csp2-H), 1664 (C=O), 1557, 1527 (Ar C=C), 1258 (C=S); 1H-NMR:
δ (ppm) 14.09 (s, 1H, COOH),
8.63 (s, 1H, Ar-H), 7.75 (s, 1H, Ar-H), 7.71 (d, 2H, ArH, J = 8.3 Hz), 7.56 (s, 1H, C=CH), 7.03 (d, 2H, ArH,
J = 8.3 Hz), 3.49–3.44 (m, 1H, CH), 3.17–3.11 (m, 4H, CH2), 2.14–2.07 (m, 4H, CH2), 2.01 (s, 3H, CH3),
1.14 (d, 4H, CH2, J = 6.3 Hz); 13C-NMR:
δ (ppm) 166.0 (acid C=O), 161.3 (amide C=O), 151.95, 141.8,
138.8, 135.7, 128.3, 124.5, 123.1, 121.6, 120.7, 118.3, 117.0, 115.6, 110.5, 107.0, 52.7, 44.0, 37.6, 9.7. Anal.
Calcd. for C23H21FN4O7S: C, 53.29; H, 3.96; N, 10.83; S, 6.21 found: C, 53.12; H, 3.84; N, 10.59; S, 6.07.
1-Cyclopropyl-7-(4-((2,3-dichlorophenyl)sulfonyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3c). White solid; yield: 79%; m.p.: 168–170 ◦C; Rf: 0.61 (1:1 petroleum ether–ethyl acetate); FTIR
1
(neat, cm−1): 3090 (Csp2-H), 1667 (C=O), 1561, 1531 (Ar C=C), 1265 (C=S), 735 (C-Cl); H-NMR:
δ
(ppm) 14.19 (s, 1H, COOH), 8.80 (s, 1H, ArH), 8.29 (s, 1H, Ar-H), 7.77 (d, 1H, Ar-H, J = 8.9 Hz), 7.53
(s, 1H, ArH), 7.34 (d, 1H, Ar-H, 8.9 Hz), 7.28 (s, 1H, C=CH), 3.59–3.55 (m, 1H, CH), 3.29–3.26 (m, 4H,

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CH2), 2.54–2.49 (m, 4H, CH2), 1.21 (d, 4H, CH2, J = 6.3 Hz); 13C-NMR:
δ
(ppm) 195.0 (ketone C=O),
171.0 (acid C=O), 169.3162.95, 151.1, 149.8, 146.7, 139.6, 134.5, 133.1, 132.6, 130.3, 129.4, 127.0, 113.6,
111.5, 52.7, 41.0, 31.6, 10.7. Anal. Calcd. for C29H26FN3O5S: C, 63.60; H, 4.78; N, 7.68; S, 5.85 found: C,
63.32; H, 4.54; N, 10.59; S, 6.07.
1-Cyclopropyl-7-(4-((3,5-dimethylphenyl)sulfonyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3d). White solid; yield: 78%; m.p.: 229–231 ◦C; Rf: 0.65 (1:1 petroleum ether–ethyl acetate); FTIR
(neat, cm-1): 3150 (Csp2-H), 1652 (C=O)„ 1593, 1551 (Ar C=C), 1475 (N=O) 1241 (C=S); 1H-NMR:
δ
(ppm): 15.09 (s, 1H, COOH), 12.40 (s, 1H, NH-C=S), 9.09 (d, 2H, Ar-H, J = 7.5 Hz), 9.04 (t, 1H, Ar-H,
J = 7.5 Hz), 8.82 (s, 1H, Ar-H), 7.95 (s, 1H, Ar-H), 7.59 (s, 1H, C=CH), 3.85–3.79 (m, 1H, CH), 3.53–3.49
(m, 4H, CH2), 3.34–3.30 (m, 4H, CH2), 2.30 (s, 6H, CH3), 1.32 (d, 4H, CH2, J = 6.5 Hz); 13C-NMR:
δ
(ppm): 192.0 (ketone C=O), 166.2 (acid C=O), 148.6, 148.3, 144.5, 144.4, 139.5, 137.1, 130.1, 129.3, 122.3,
119.8, 119.7, 111.7, 111.4, 107.4, 46.9, 46.8, 43.1, 36.4, 21.6, 8.1. Anal. Calcd. for C23H20Cl2FN3O5S: C,
51.14; H, 3.76; N, 7.76; S, 5.91 found: C, 50.92; H, 3.64; N, 7.59; S, 6.07.
1-Cyclopropyl-6-fluoro-7-(4-((4-methyl-3-nitrophenyl)sulfonyl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-
◦
carboxylic acid (3e). White solid; yield: 80%; m.p.: 160–162 C; Rf: 0.61 (1:1 petroleum ether–ethyl
acetate); FTIR (neat, cm−1): 3030 (Csp2-H), 1664 (C=O), 1557, 1527 (Ar C=C), 1258 (C=S); 1H-NMR:
δ
(ppm) 14.09 (s, 1H, COOH), 8.63–7.71 (m, 3H, Ar-H), 7.56 (s, 1H, C=CH), 7.03 (d, 2H, ArH, J = 8.3 Hz),
3.49–3.44 (m, 1H, CH), 3.17–3.11 (m, 4H, CH2), 2.14–2.07 (m, 4H, CH2), 2.01 (s, 3H, CH3), 1.14 (d, 4H,
CH2, J = 6.3 Hz); 13C-NMR:
δ (ppm) 190.0 (ketone C=O), 166.0 (acid C=O), 151.95, 138.8, 135.7,
128.3, 124.5, 123.1, 121.6, 120.7, 118.3, 117.0, 115.6, 110.5, 107.0, 52.7, 44.0, 37.6, 9.7. Anal. Calcd. for
C23H20Cl2FN3O5S: C, 51.14; H, 3.76; N, 7.76; S, 5.91 found: C, 50.92; H, 3.64; N, 7.59; S, 6.07.
0
7-(4-([1,1 -Biphenyl]-4-ylsulfonyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3f). White solid; yield: 80%; m.p.: 160–163 ◦C; Rf: 0.63 (1:1 petroleum ether–ethyl acetate); FTIR
(pure, cm−1): 3062 (Csp2-H), 1667 (C=O), 1558, 1531 (Ar C=C), 1248 (C=S), 1H-NMR:
δ
(ppm) 14.29
(s, 1H, COOH), 8.75 (s, 1H, Ar-H), 7.91 (s, 1H, Ar-H), 7.83 (d, 2H, ArH, J = 8.5 Hz), 7.51 (s, 1H, C=CH),
7.43 (d, 2H, ArH, J = 8.5 Hz), 7.40–6.92 (m, 5H, ArH), 3.65–3.61 (m, 1H, CH), 3.18–3.11 (m, 4H, CH2),
2.74–3.67 (m, 4H, CH2), 1.22 (d, 4H, CH2, J = 6.1 Hz); 13C-NMR: δ (ppm) 196.0 (C=O of ketone), 170.0
(C=O acid), 150.8, 149.8, 14677, 139.3, 138.5, 137.1, 135.6, 133.7, 129.3, 135.1, 133.6, 131.7, 129.3, 128.0,
117.6, 111.5, 53.7, 45.0, 39.6, 11.7. Anal. Calcd. for C24H23FN4O7S: C, 54.34; H, 4.36; N, 10.55; S, 5.99
found: C, 54.12; H, 3.98; N, 10.15; S, 6.07.
4-Methyl-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)benzenesulfonamide (6a). White solid; yield: 81%; m.p.:
223–225 ◦C; Rf: 0.63 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3260 (NH) 3133 (Csp2-H),
1
1587, 1540 (Ar C=C), H-NMR:
δ (ppm) 8.08 (d, 2H, J =4.8 Hz, pyrimidyl), 7.09 (t, 1H, J = 4.8 Hz,
pyrimidinyl), 7.38 (d, 2H, J = 8.4 Hz, C6H4-meta), 7.86 (d, 2H, J = 8.4 Hz, C6H4-ortho), 7.18 (d, 2H,
J =8.4 Hz, C6H4-meta), 7.06 (d, 2H, J = 8.4 Hz, C6H4-ortho) 9.27 (s, 1H, NH) 2.21 (s, 3H, CH3), 13C-NMR:
δ
(ppm) 160.5 (C=N), 158.4, 141.8, 138.8, 135.7, 133.2, 132.5, 130.5, 129.5, 128.3, 120.5, 26.6. Anal. Calcd.
For C17H16N4O4S2: C, 50.47; H, 3.99; N, 13.84; S, 15.85 found: C, 50.12; H, 3.78; N, 13.55; S, 16.67.
2-Nitro-N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)benzenesulfonamide (6b). White solid; yield: 80%; m.p.:
160–162 ◦C; Rf: 0.61 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3260 (NH) 3131 (Csp2-H),
1586, 1544 (Ar C=C), 1254 (C=S); 1H-NMR:
δ
(ppm) 8.08 (d, 2H, J = 4.8 Hz, pyrimidyl), 7.09 (t, 1H,
J = 4.8 Hz, pyrimidinyl), 7.38 (d, 2H, J =8.4 Hz, C6H4-meta), 8.26 (d, 2H, J = 8.4 Hz, C6H4-ortho), 7.38
d, 2H, J = 8.4 Hz, C6H4-meta), 7.06 (d, 2H, J = 8.4 Hz, C6H4-ortho), 9.30 (s, 1H, NH) 13C-NMR:
(ppm)
(
δ
181.5 (C=S), 142.8, 139.8, 135.8, 127.3. Anal. Calcd. for C17H16N4O4S2: C, 44.12; H, 3.02; N, 16.07; S,
14.75 found: C, 43.98; H, 2.99; N, 15.98; S, 14.61.
0
N-(4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)-[1,1 -biphenyl]-4-sulfonamide (6c). White solid; yield: 79%; m.p.:
168–170 ◦C; Rf: 0.51 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3263 (NH) 3132 (Csp2-H),
1576, 1542 (Ar C=C), 1253 (C=S); 1H-NMR:
δ
(ppm) 8.18 (d, 2H, J = 4.8 Hz, pyrimidyl), 7.19 (t, 1H,
J = 4.8 Hz, pyrimidinyl), 7.82 (d, 2H, J =8.4 Hz, C6H4-meta), 7.76 (d, 2H, J = 8.4 Hz, C6H4-ortho), 7.22

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(
d, 2H, J = 8.4 Hz, C6H4-meta), 7.16 (d, 2H, J = 8.4 Hz, C6H4-ortho), 7.75–7.39 (m, 5H, Ar), 9.37 (s, 1H,
NH) 13C-NMR:
δ
(ppm) 186.5 (C=S), 142.8, 141.7, 139.8, 138.5, 135.8, 133.5, 132.5, 127.3. Anal. Calcd.
for C22H18N4O4S2: C, 56.12; H, 3.89; N, 12.03; S, 13.73 found: C, 55.98; H, 3.76; N, 11.98; S, 13.61.
2-((2,3-Dichlorophenyl)sulfonyl)hydrazine-1-carbothioamide (6d). White solid; yield: 70%; m.p.: 223–225 ◦C
;
Rf: 0.63 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3260 (NH) 3133 (Csp2-H), 1587, 1540
(
Ar C=C), 1H-NMR:
7.38 (d, 2H, J = 8.4 Hz, C6H4-meta), 7.86 (d, 2H, J = 8.4 Hz, C6H4-ortho), 7.18 (d, 2H, J = 8.4 Hz,
C6H4-meta), 7.06 (d, 2H, J = 8.4 Hz, C6H4-ortho) 9.27 (s, 1H, NH) 2.21 (s, 3H, CH3), 13C-NMR:
(ppm)
δ (ppm) 8.08 (d, 2H, J = 4.8 Hz, pyrimidyl), 7.09 (t, 1H, J = 4.8 Hz, pyrimidinyl),
δ
179.5 (C=S), 141.8, 138.8, 135.7, 128.3, 26.6. Anal. Calcd. For C16H12 Cl2N4O4S2: C, 41.83; H, 2.64; N,
12.19; S, 13.93 found: C, 41.48; H, 2.46; N, 11.98; S, 13.61.
2-((3,5-Dimethylphenyl)sulfonyl)hydrazine-1-carbothioamide (6e). White solid; yield: 76%; m.p.:
223–225 ◦C; Rf: 0.63 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3260 (NH) 3133 (Csp2-H),
1
1587, 1540 (Ar C=C), H-NMR:
δ (ppm) 8.08 (d, 2H, J = 4.8 Hz, pyrimidyl), 7.09 (t, 1H, J = 4.8 Hz,
pyrimidinyl), 7.38 (d, 2H, J = 8.4 Hz, C6H4-meta), 7.86 (d, 2H, J = 8.4 Hz, C6H4-ortho), 7.18 (d, 2H,
J = 8.4 Hz, C6H4-meta), 7.06 (d, 2H, J = 8.4 Hz, C6H4-ortho) 9.27 (s, 1H, NH) 2.21 (s, 3H, CH3), 13C-NMR:
δ
(ppm) 179.5 (C=S), 141.8, 138.8, 135.7, 128.3, 26.6. Anal. Calcd. For C18H18 N4O4S2: C, 51.53; H, 4.54;
N, 13.39; S, 15.32 found: C, 51.35; H, 4.36; N, 13.23; S, 15.12.
2-((4-Methyl-3-nitrophenyl)sulfonyl)hydrazine-1-carbothioamide (6f). White solid; yield: 70%; m.p.:
223–225 ◦C; Rf: 0.63 (1:1 petroleum ether: ethyl acetate); FTIR (neat, cm−1): 3260 (NH) 3133 (Csp2-H),
1587, 1540 (Ar C=C), 1H-NMR: 8.08 (d, 2H, J = 4.8 Hz, pyrimidyl), 7.09 (t, 1H, J = 4.8 Hz, pyrimidinyl),
7.38 (d, 2H, J = 8.4 Hz, C6H4-meta), 7.86 (d, 2H, J = 8.4 Hz, C6H4-ortho), 7.18 (d, 2H, J = 8.4 Hz,
C6H4-meta), 7.06 (d, 2H, J = 8.4 Hz, C6H4-ortho) 9.27 (s, 1H, NH) 2.21 (s, 3H, CH3), 13C-NMR:
δ (ppm)
179.5 (C=S), 141.8, 138.8, 135.7, 128.3, 26.6. Anal. Calcd. for C18H18 N4O4S2: C, 51.49; H, 4.44; N, 13.29;
S, 14.32 found: C, 51.35; H, 4.31; N, 13.21; S, 14.99.
N-(Adamantan-1-yl)-4-methylbenzenesulfonamide (9a). White solid; yield: 73%; m.p.: 218–222◦C; Rf: 0.61
(1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3262 (NH) 3134 (Csp2-H), 1587, 1544 (Ar C=C),
1H-NMR:
δ
(ppm) 7.38 (d, 2H, J = 8.4 Hz, C6H4-meta), 7.86 (d, 2H, J = 8.4 Hz, C6H4-ortho), 9.27 (s, 1H,
NH) 2.21 (s, 3H, CH3), 1.67–1.79 (m, 6H, CH2); 1.85 (s, 6H, CH2); 2.29 (s, 3H, CH) 13C-NMR:
δ
(ppm)
141.75, 137.74, 130.49, 129.49, 40.98, 35.80, 29.74, 29.12, 22.4. Anal. Calcd. for C17H23NO2S: C, 66.84; H,
7.58; N, 4.56; S, 10.51 found: C, 66.67; H, 7.41; N, 4.31; S, 10.40.
N-(Adamantan-1-yl)-4-nitrobenzenesulfonamide (9b). White solid; yield: 84%; m.p.: 164–168 ◦C; Rf: 0.63
(1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3263 (NH) 3131 (Csp2-H), 1584, 1543 (Ar C=C),
1H-NMR:
δ
(ppm) 7.48 (d, 2H, J = 8.4 Hz, C6H4-meta), 8.22 (d, 2H, J = 8.4 Hz, C6H4-ortho), 9.30 (s, 1H,
NH) 2.24 (s, 3H, CH3), 1.65–1.77 (m, 6H, CH2); 1.84 (s, 6H, CH2) 13C-NMR:
δ
(ppm) 143.75, 136.74,
131.49, 128.49, 40.99, 35.85, 29.72, 29.123. Anal. Calcd. for C16H20N2O4S: C, 57.11; H, 5.98; N, 8.32; S,
9.52 found: C, 56.99; H, 5.81; N, 7.98; S, 9.41.
0
◦
N-(Adamantan-1-yl)-[1,1 -biphenyl]-4-sulfonamide (9c). White solid; yield: 76%; m.p.: 165–171 C; Rf: 0.55
(1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3263 (NH) 3132 (Csp2-H), 1576, 1542 (Ar C=C),
1H-NMR:
δ
(ppm) 7.82 (d, 2H, J =8.4 Hz, C6H4-meta), 7.76 (d, 2H, J = 8.4 Hz, C6H4-ortho), 7.75–7.39
(m, 5H, Ar), 9.37 (s, 1H, NH), 2.22 (s, 3H, CH3), 1.64–1.76 (m, 6H, CH2); 1.82 (s, 6H, CH2) 13C-NMR:
δ
(ppm) 142.8, 141.7, 139.8, 138.5, 135.8, 133.5, 132.5, 127.3, 41.91, 36.82, 29.71, 28.12. Anal. Calcd. for
C18H21NO2S: C, 68.54; H, 6.73; N, 4.45; S, 10.18 found: C, 68.43; H, 5.81; N, 4.28; S, 9.97.
◦
N-(Adamantan-1-yl)-2,3-dichlorobenzenesulfonamide (9d). White solid; yield: 78%; m.p.: 221–230 C;
Rf: 0.61 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3255 (NH) 3135 (Csp2-H), 1580, 1542
1
(
Ar C=C), H-NMR:
δ
(ppm) 6.64–7.97 (m, 3H, ArH) 9.24 (s, 1H, NH) 2.23 (s, 3H, CH3), 1.54–1.66
(m, 6H, CH2); 1.72 (s, 6H, CH2); 13C-NMR: δ (ppm) 142.8, 139.8, 135.8, 133.5, 131.4, 127.3, 42.91, 38.82,

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30.71, 27.12. Anal. Calcd. for C16H19Cl2NO2S: C, 53.35; H, 5.32; N, 3.87; S, 8.18 found: C, 53.13; H, 5.12;
N, 3.78; S, 8.99.
◦
N-(Adamantan-1-yl)-3,5-dimethylbenzenesulfonamide (9e). White solid; yield: 81%; m.p.: 162–164 C;
Rf: 0.61 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3248 (NH) 3156 (Csp2-H), 1572, 1532
(
Ar C=C), 1H-NMR:
δ
(ppm) 7.61–7.97 (m, 3H, ArH) 9.34 (s, 1H, NH); 2.36 (s, 6H, CH3), 2.21 (s, 3H,
CH3), 1.58–1.68 (m, 6H, CH2), 1.73 (s, 6H, CH2); 13C-NMR:
δ
(ppm) 141.8, 139.8, 134.8, 127.3, 43.91,
39.82, 31.71, 29.12 27.5. Anal. Calcd. for C18H25NO2S: C, 67.65; H, 7.88; N, 4.38; S, 10.04 found: C, 67.45;
H, 7.69; N, 4.09; S, 9.92.
N-(Adamantan-1-yl)-4-methyl-3-nitrobenzenesulfonamide (9f). White solid; yield: 80%; m.p.: 165–163 ◦C;
Rf: 0.61 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3251 (NH) 3152 (Csp2-H), 1567, 1537
(Ar C=C), 1H-NMR:
δ
(ppm) 8.10–7.75 (m, 3H, ArH) 9.72 (s, 1H, NH) 2.38 (s, 3H, CH3), 2.23 (s, 3H,
CH3), 1.56–1.64 (m, 6H, CH2); 1.72 (s, 6H, CH2); 13C-NMR:
δ
(ppm) 144.8, 137.8, 136.8, 131.3, 128.5,
126.5 23.6, 47.91, 43.82, 36.71, 28.12. Anal. Calcd. for C17H22N2O4S: C, 58.28; H, 6.34; N, 7.98; S, 9.14
found: C, 58.14; H, 5.97; N, 4.09; S, 8.98.
2-Tosylhydrazine-1-carbothioamide (12a). White solid; yield (76%; m.p.: 220–222 ◦C; Rf: 0.63
(
1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3264 (NH) 3135 (Csp2-H), 1589, 1541 (Ar C=C),
1251 (C=S); 1H-NMR:
9.27 (s, 1H, NH) 9.31 (s, 1H, NH) 7.1 (s, 2H, -NH2); 2.21 (s, 3H, CH3); 13C-NMR:
δ
(ppm) 7.38 (d, 2H, J = 8.4 Hz, C6H4-meta), 7.86 (d, 2H, J = 8.4 Hz, C6H4-ortho),
(ppm) 179.5 (C=S),
δ
141.8, 138.8, 135.7, 128.3, 26.6. Anal. Calcd. For C18H11N2O4S: C, 45.07; H, 5.21; N, 19.71; S, 15.04 found:
C, 44.94; H, 4.97; N, 19.49; S, 14.97.
2-((4-Nitrophenyl)sulfonyl)hydrazine-1-carbothioamide (12b). White solid; yield: 80%; m.p.: 160–162 ◦C;
Rf: 0.61 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3260 (NH) 3131 (Csp2-H), 1586, 1544
(Ar C=C), 1254 (C=S); 1H-NMR:
δ
(ppm) 7.38 (d, 2H, J = 8.4 Hz, C6H4-meta), 8.26 (d, 2H, J = 8.4 Hz,
C6H4-ortho), 9.30 (s, 1H, NH) 9.29 (s, 1H, NH) 7.1 (s, 2H, -NH2); 13C-NMR:
δ
(ppm) 181.5 (C=S), 142.8,
139.8, 135.8, 127.3. Anal. Calcd. for C7H8N4O4S2: C, 30.42; H, 2.91; N, 20.29; S, 23.21 found: C, 30.12;
H, 2.82; N, 20.13; S, 23.11.
0
2-([1,1 -Biphenyl]-4-ylsulfonyl)hydrazine-1-carbothioamide (12c). White solid; yield: 79%; m.p.: 168–170 ◦C
Rf: 0.51 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3263 (NH) 3132 (Csp2-H), 1576, 1542
(Ar C=C), 1253 (C=S); 1H-NMR:
(ppm) 7.82 (d, 2H, J = 8.4 Hz, C6H4-meta), 7.76 (d, 2H, J = 8.4 Hz,
C6H4-ortho), 7.75–7.39 (m, 5H, Ar), 9.37 (s, 1H, NH), 9.28 (s, 1H, NH), 7.5 (s, 2H, -NH2); 13C-NMR:
;
δ
δ
(ppm) 186.5 (C=S), 142.8, 141.7, 139.8, 138.5, 135.8, 133.5, 132.5, 127.3. Anal. Calcd. for C13H13N3O2S2:
C, 50.79; H, 4.27; N, 13.65; S, 20.21 found: C, 50.57; H, 4.12; N, 13.19; S, 19.07.
2-((2,3-Dichlorophenyl)sulfonyl)hydrazine-1-carbothioamide (12d). White solid; yield: 78%; m.p.:
229–231 ◦C; Rf: 0.65 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3258 (NH) 3137 (Csp2-H),
1
1582, 1540 (Ar C=C), 1264 (C=S); H-NMR:
δ
(ppm) 6.64–7.97 (m, 3H, ArH) 9.22 (s, 1H, NH), 9.17
(s, 1H, NH), 7.43 (s, 2H, -NH2); 13C-NMR:
δ (ppm) 187.5 (C=S), 142.8, 139.8, 135.8, 133.5, 131.4, 127.3.
Anal. Calcd. for C7H7Cl2N3O2S2: C, 28.79; H, 4.27; N, 13.65; S, 20.21 found: C, 50.57; H, 4.12; N,
13.19; S, 19.07.
2-((3,5-Dimethylphenyl)sulfonyl)hydrazine-1-carbothioamide (12e). White solid; yield: 80%; m.p.: 160–162
◦C; Rf: 0.61 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3258 (NH) 3156 (C-H), 1578, 1534
(Ar C=C), 1250 (C=S); 1H-NMR:
(s, 2H, -NH2), 2.36 (s, 6H, CH3); 13C-NMR:
Calcd. for C9H13N3O2S2: C, 41.68; H, 5.06; N, 16.20; S, 24.71 found: C, 41.49; H, 4.90; N, 15.96; S, 24.59
δ
(ppm) 7.61–7.97 (m, 3H, ArH), 9.34 (s, 1H, NH), 9.28 (s, 1H, NH), 7.76
(ppm) 181.5 (C=S), 141.8, 139.8, 134.8, 127.3, 27.5. Anal.
δ
.
2-((4-Me◦thyl-3-nitrophenyl)sulfonyl)hydrazine-1-carbothioamide (12f). White solid; yield: 80%; m.p.:
160–163 C; Rf: 0.63 (1:1 petroleum ether–ethyl acetate); FTIR (neat, cm−1): 3252 (NH) 3150 (C-H), 1568,
1539 (Ar C=C), 1253 (C=S); 1H-NMR:
7.73 (s, 2H, -NH2), 2.38 (s, 3H, CH3); 13C-NMR:
δ
(ppm) 8.11–7.77 (m, 3H, ArH) 9.74 (s, 1H, NH) 9.68 (s, 1H, NH),
(ppm) 171.5 (C=S), 145.8, 138.8, 137.8, 130.3, 129.5,
δ

Molecules 2017, 22, 1352
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127.5 24.6. Anal. Calcd. for C8H10N4O4S2: C, 33.10; H, 3.47; N, 19.30; S, 22.09 found: C, 32.59; H, 3.32;
N, 19.16; S, 23.98.
4.4. Urease Inhibition Assay
The jack bean urease action was determined by measuring the ammonia produced by the
indophenol method described by Weatherburn. The reaction mixtures, containing 50
(100 mM urea, 10 mM dibasic potassium phosphate, 1 mM EDTA and 10 mM LiCl, pH 8.2), 20
sample and 20 L of the jack bean urease (5 U/mL) were added in a 96-well plate and the plate was
incubated at 37 ◦C for 30 min. After incubation, 50
L of phenol reagent (1%, w/v phenol and 0.005%,
µL of buffer
µL of
µ
µ
w/v sodium nitroprusside) and 50
µ
L of alkali reagents (0.5%, w/v NaOH and 0.1% NaOCl) were
◦
added in each well, and again the plate was incubated at 37 C for 10 min. After the second incubation,
the absorbance was checked at 625 nm, using a microplate reader (OPTIMax, Tunable, Sunnyvale,
CA, USA). All reactions were performed in triplicate. The urease inhibition activity was calculated
according to the following formula:
Inhibition (%) = [(B − S)/B] × 100
where B and S are the optical densities of sample and blank. Thiourea was used as reference.
4.5. Kinetic Mechanism Study
Kinetic analysis was carried out to determine the mode of inhibition. Four inhibitors (3e, 6a, 9e
and 12d) were selected on the basis of the most potent IC50 values. The most potent compound was
selected from each group. Kinetics were determined by varying the dose of urea in the presence of
different doses of compound 3e (0.025, 0.05, 0.01 and 0.02
and 0.0044 M), compound 9e (0.00, 0.015, 0.03 and 0.06
0.052, 0.104 and 0.208 M). In detail the urea doses were varied between 100, 50, 25, 12.5, 6.25 and
µM), compound 6a (0.00, 0.0011, 0.0022,
µ
µM) and compound 12d (0.0130, 0.026,
µ
3.125 mM mM for urease kinetics studies and the rest of the procedure was same for all kinetic studies
as described in the urease inhibition assay protocol. Maximal initial velocities were determined from
the initial linear portion of the absorbances up to 10 min after the addition of enzyme at per minute’s
intervals. The inhibition type of the enzyme was assayed by Lineweaver–Burk plots of the inverse
of velocities (1/V) versus the inverse of substrate concentration 1/[S] mM−1. The EI dissociation
constant Ki was determined by the secondary plot of 1/V versus inhibitor concentration and the ESI
dissociation constant Ki0 was calculated from the intercept versus inhibitor concentrations. Urease
activity was determined by measuring ammonia production using the indophenol method as reported
previously [50]. The results (change in absorbance per min) were recorded at 625 nm using a microplate
reader (Optimax Tunable, Sunnyvale, CA, USA).
4.6. Free Radical Scavenging Assay
Radical scavenging activity was determined by adjusting the already reported 2,2-diphenyl-1-
picrylhydrazyl (DPPH) assay method [51]. The assay arrangement comprised 100
µL of DPPH
(150 M), 20 L of increasing concentrations of test compounds and the volume was adjusted to 200 µL
µ
µ
in each well with DMSO. At a room temperature, the reaction mixture was incubated for 30 min.
Ascorbic acid (Vitamin C) was utilized as a model inhibitor. The assay estimations were done by using
a micro plate reader (OPTIMax, Tunable, Sunnyvale, CA, USA) at 517 nm. The response rates were
analyzed and the percent inhibition caused by the presence of the tested inhibitor was calculated. Each
concentration was analyzed in three independent experiments run thrice.
4.7. Retrieval of Jack Bean Urease Structure from Protein Data Bank (PDB)
The crystal structure of jack bean urease (PDBID 4H9M) was retrieved from the Protein Data Bank
(PDB, www.rcsb.org). The selected protein structure was minimized by employing the University of

Molecules 2017, 22, 1352
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California at San Francisco (UCSF) Chimera 1.10.1 tool [52]. Furthermore, the stereochemical properties
of the urease structure and Ramachandran plot and values were generated by Molprobity server [53].
The Discovery Studio 4.1 Client tool was employed to generate the hydrophobicity graph of the target
protein [54]. The protein architecture and statistical percentage values of urease helices,
and turn were predicted using the online server VADAR 1.8 [55].
β-sheets, coils
4.8. Molecular Docking of Synthesized Compounds Using AutoDock
Molecular docking experiments were done for all the synthesized ligands 3a
–f, 6a–f, 9a–f and
12a–f against urease using different AutoDock 4.2 tools according to the specified instructions [56].
In the receptor (urease) the polar hydrogen atoms and Kollman charges were apportioned and for
the ligands, 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 the receptor as rigid, whereas the ligands
were considered as flexible molecules. Grid map values were adjusted at 80 Å
×
80 Å
×
80 Å on the
targeted protein to generate the grid map. 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 default docking parameters. 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, (Accelrys, San Diego, CA, USA, 4.1)
and UCSF Chimera 1.10.1.
4.9. Molecular Dynamics Simulations Assay
Based on in vitro analysis, docking energy and conformational pose analysis the four best
complexes were selected for molecular dynamic simulations. Groningen Machine for Chemicals
Simulations (GROMACS) 4.5.4 package [57] with the GROMOS 53A6 force field [58] were applied on
all complexes to interpret the backbone residual flexibility. The receptor and ligands topology files
were created by using the GROMOS 53A6 force-field and the online PRODRG Server [59], respectively.
Moreover, the receptor–ligand complexes were solvated and placed in the center of a cubic box
adjusted to 9 Å distance. Ions were added to neutralize the system charges. Energy minimization
(nsteps = 50,000) was done by the steepest descent method (1000 ps). The energy calculation was done
by the Particle Mesh Ewald (PME) method [60], whereas covalent bond constraints were calculated
by the linear constraint solver (LINCS) algorithm [61]. For final molecular dynamics (MD) setup in a
md.mdp file, the time step for integration was adjusted as 0.002 ps, while the cutoff distance for short
range neighbor list (rlist) was adjusted to 0.8 nm. The MD run was set to 10,000 ps with nsteps 500,000
for each protein-ligand complexes and trajectories files analysis was done by the Xmgrace tool [47].
5. Conclusions
A small library of compounds based on drug derivatives was synthesized using substituted
sulphonyl chlorides. All the synthesized compounds were evaluated for jack bean urease inhibition and
free radical scavenging activity. Interestingly, all the drug derivatives elicited superior activity against
urease enzyme compared to the standard used (thiourea). The kinetic mechanism was determined for
the most potent derivatives and it was found that compounds 3e, 6a, and 12d exhibited a mixed type of
mode inhibition, while compound 9e inhibited urease via a non-competitive mechanism. The enzyme
inhibition (Ki) value was found to be in the micromolar range, which further ascertained the efficacy of
the synthesized ligands against the target enzyme. In addition to this, molecular docking studies were
performed to explore the non-covalent interactions and chemoinformatics data were also evaluated
using the rule of five or Lipinski’s rule and most of the compounds were found to be in good agreement
with these rules. Extensive bioinformatics studies were carried out. From the results we conclude that
these compounds could serve as lead compounds in designing structural templates for the inhibition

Molecules 2017, 22, 1352
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of urease enzyme. This is also proof of the concept that simple manipulations of known drugs can led
to the discovery of new drugs.
Supplementary Materials: Supplementary Materials are available online.
Acknowledgments: This work was funded in part by the following: the National Research Foundation (NRF)
and the University of KwaZulu-Natal (South Africa); and the CICYT (CTQ2015-67870-P), and the Generalitat de
Catalunya (2014 SGR 137) (Spain).
Author Contributions: Pervaiz Ali Channar and Aamer Saeed conceived and designed the experiments;
Qamar Abbas and Hussain Raza performed the experiments; and Fernando Albericio and Sung-Yum Seo analyzed
the data; Mubashir Hassan contributed reagents/materials/analysis tools; Fayaz Ali Larik wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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