Developing new hybrid scaffold for urease inhibition based on carbazole-chalcone conjugates: Synthesis, assessment of therapeutic potential and computational docking analysis

Article abstract of DOI:10.1016/j.bmc.2019.115123

Although a diverse range of chemical entities offering striking therapeutic potential against urease enzyme has been reported, the key challenges (toxicity and safety) associated with these inhibitors create a large unmet medical need to unveil new, poten

Full text of DOI:10.1016/j.bmc.2019.115123

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Developing new hybrid scaffold for urease inhibition based on
carbazole-chalcone conjugates: Synthesis, assessment of
therapeutic potential and computational docking analysis
Madiha Kazmi, Imtiaz Khan, Ajmal Khan, Sobia Ahsan Halim,
Aamer Saeed, Saifullah Mehsud, Ahmed Al-Harrasi, Aliya Ibrar
PII:
S0968-0896(19)31109-5
https://doi.org/10.1016/j.bmc.2019.115123
BMC 115123
DOI:
Reference:
To appear in:
Received date:
Revised date:
Accepted date:
29 June 2019
11 September 2019
14 September 2019
Please cite this article as: M. Kazmi, I. Khan, A. Khan, et al., Developing new hybrid
scaffold for urease inhibition based on carbazole-chalcone conjugates: Synthesis,
assessment of therapeutic potential and computational docking analysis, (2019),
https://doi.org/10.1016/j.bmc.2019.115123
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Developing new hybrid scaffold for urease inhibition based on carbazole-chalcone conjugates:
Synthesis, assessment of therapeutic potential and computational docking analysis
a,b,†
c,d, †
e
e
a,
f
Madiha Kazmi,
Imtiaz Khan,
Ajmal Khan, Sobia Ahsan Halim, Aamer Saeed, * Saifullah Mehsud,
e,
a,f,
Ahmed Al-Harrasi, * Aliya Ibrar, *
Department of Chemistry, Quaid‒i‒Azam University, Islamabad 45320, Pakistan
a
b
Department of Chemistry, University of Gujrat, Rawalpindi Sub‒campus, Satellite Town, Rawalpindi,
Pakistan
c
School of Chemistry, The University of Manchester, Oxford Road, Manchester M13 9PL, United Kingdom
d
Manchester Institute of Biotechnology, The University of Manchester, 131 Princess Street, Manchester M1
7
DN, United Kingdom
e
Natural and Medical Sciences Research Center, University of Nizwa, P.O Box 33, Postal Code 616, Birkat
Al Mauz, Nizwa, Sultanate of Oman
f
Department of Chemistry, Abbottabad University of Science and Technology, Havelian, Abbottabad,
Pakistan
†
These authors contributed equally to this work.
Correspondence: jadoonquaidian@gmail.com (A. Ibrar); aharrasi@unizwa.edu.om (A. Al-Harrasi);
aamersaeed@yahoo.com (A. Saeed)
Abstract
Although a diverse range of chemical entities offering striking therapeutic potential against urease enzyme
has been reported, the key challenges (toxicity and safety) associated with these inhibitors create a large
unmet medical need to unveil new, potent and safe inhibitors of urease enzyme. In this pursuit, the present
study demonstrates the successful synthesis of carbazole-chalcone hybrids (4a-n) in good yields. The
evaluation of the preliminary in vitro biological results showed that selected members of the investigated
library of hybrid compounds possess excellent urease inhibitory efficacy. In particular, compounds 4c and 4k
were the most potent inhibitors with lowest IC50 values of 8.93 ± 0.21 and 6.88 ± 0.42 µM, respectively.
Molecular docking analysis of the most potent inhibitor 4k suggests that the compound is fitted neatly at the

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active site interface and mediates interaction with both nickel atoms present in the active site. Several other
obvious interactions including metal-carbonyl contact, hydrogen bonding and hydrophobic interactions were
also observed, playing a crucial part in the stabilization of 4k in the active site of urease.
Keywords: Carbazole; Chalcone; Hybrid scaffold; Urease; Structural diversity; Enzyme inhibition.
1
. Introduction
Despite many technological advances, the identification, optimization and selection of a candidate drug
molecule with desirable properties have remained a challenging task, thus appealing medicinal chemists to
explore the wider chemical space in the hunt for effective therapeutics. Notably, this drug discovery
compaign is well-driven by nitrogen-containing heterocycles which make the significant structural
components of numerous pharmaceuticals and natural products [1-4]. Among N-heterocycles, carbazoles
constitute an important class of fused aromatics with established profile of biological significance. They are
widely encountered as privileged motifs in numerous bioactive natural products and pharmaceuticals [5-10].
Carbazole and its derivatives have successfully demonstrated their biological potential in terms of their
antitumor, antimicrobial, antihistaminic, antioxidative, anti-inflammatory, and psychotropic [7,11-15]
activities in addition to their application as useful building blocks for the synthesis of functional organic
materials [16,17].
In a similar fashion to carbazoles, chalcones (α,β,-unsaturated ketones) represent an important class of open
chain flavonoids with a diverse range of applications in synthetic and medicinal chemistry [18]. The easy
accessibility of these molecules offers a convenient platform to exploit their potential to serve as new leads in
drug discovery [19,20]. The enriching literature successfully demonstrate the diverse use of natural and
synthetic chalcones in the form of various biological activities such as anticancer [21], anti-inflammatory
[22], antioxidant [23], antimicrobial [24], antimalarial [25], anti-HIV [26], anti-arrhythmic [27], antiplatelet
[28], anti-diabetic [29], anti-neoplastic [30], anti-angiogenic [31], anti-retroviral [32], and monoamine
oxidase inhibitors [33], among many others [34-37]. In addition, several hybrid chalcones incorporating
heterocyclic systems [38,39], biphenyl functionality [40] or other substitutions [41-43] have also displayed

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anticancer and antiviral activities [44,45]. Figure 1 represents the illustrative bioactive potential of carbazole
and chalcone scaffolds [46,47].
Fig. 1. Selected examples of carbazole and chalcones as useful bioactive motifs.
Urease (EC 3.5.1.5), a nickel-containing heteropolymeric amidohydrolase, is responsible for the hydrolysis
of urea to ammonia and carbon dioxide [48]. It is abundantly present in fungi, bacteria, plants and
vertebrates. The active site of urease contains two nickel ions coordinated to four histidine residues,
carbamoylated lysine and an aspartate carboxylate [49]. This well-recognized enzyme is heavily involved in
the rapid increase of ammonia level, thus providing a sustainable environment for the survival of
Helicobacter pylori in the stomach [50]. This colonization of pathogenic microorganisms causes several
urinary and gastrointestinal tract infections [51]. In agriculture, the efficacy of nitrogen fertilizers can be
decreased by soil macrobiotic ureases [52] which require heavy use of urea and causes water eutrophication
[
53]. Several classes of structurally diverse compounds have been documented to regulate the urease activity.
For instance, hydroxamic acid derivatives [54], phosphorodiamidates [55], imidazoles [56], phosphonates
57], phosphinates [58], urea and thiourea derivatives [59], triazoles, thiadiazoles [60], coumarins [61],
semicarbazones [62], Schiff bases [63], oxadiazoles [64], piperidines [65], thiols [66], ebselen derivatives
67] and metal ions [68]. However, due to severe side effects and toxicity [69] associated with these
[
[
inhibitors, there remained a suitable and unmet therapeutic need to unveil new, potent and safe inhibitors of
urease enzyme addressing the key issues highlighted in pharmaceutical research and agriculture.
Examples described herein clearly demonstrate the medicinal chemistry value of carbazole and chalcone
motifs. To combat several pathological conditions associated with ureolytic enzyme (urease), and to increase

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the size of structural libraries of new inhibitors with broadened diversity, we herein report the synthesis of
carbazole-chalcone hybrids as a new combined template to function as novel urease inhibitors. This
integrated approach combining heterocyclic and non-heterocyclic fragments would play a guiding role in the
generation of new molecules with robust therapeutic potential against urease enzyme. In addition, the in vitro
inhibitory results produced by these chemical scaffolds were rationalized using molecular docking tools.
2
2
. Results and Discussion
.1. Synthetic chemistry
The synthetic route adopted to access a small library of carbazole-chalcone hybrids is depicted in Scheme 1.
N-alkylation of carbazole 1 was achieved with octyl bromide in the presence of KOH and catalytic amount
(2.5 mol%) of n-tetrabutylammonium bromide (TBAB) under reflux conditions [70]. The formation of N-
octylcarbazole was confirmed by its FTIR spectrum where characteristic stretching bands for Csp3–H
–
1
–1
1
appeared at 2846 cm alongside the disappearance of NH band around 3400 cm . H NMR spectral data
revealed the formation of N-octylcarbazole by the disappearance of NH proton of carbazole moiety and by
1
3
the appearance of aliphatic protons in the range of 4.41‒0.86 ppm. C NMR spectrum also confirmed the
formation of N-octylcarbazole 2 as the signal appeared at 63.5‒24.3 ppm for aliphatic carbons of octyl chain.
Friedal-Crafts acylation of N-octyl carbazole 2 with acetyl chloride (2.0 equiv) afforded the diacylated
carbazole 3 in 92% yield. FTIR spectroscopy confirmed the acetylation of N-octylcarbazole by the
–
1
1
appearance of characteristic ketone carbonyl (C=O) peak at 1710 cm . In H NMR, methyl protons at 2.79
1
3
ppm confirmed the diacetylated product 3. C NMR further confirmed the acetylation, where carbonyl
(C=O) carbon appeared at 197.8 ppm. Good quality crystals were grown at room temperature using ethanol
and molecular structure of 3 was confirmed through X-ray crystallography [71]. Aldol condensation of 3
with appropriate aldehydes (1.0 equiv) afforded the desired carbazole-chalcones (4a‒n) under basic
conditions at room temperature [70]. The title products were isolated in good yields (59‒73%). A diverse
range of electron-rich, electron-deficient aryl as well as hetero-aryl aldehydes was well tolerated. The
formation of desired hybrid structures (4a‒n) was recognized by FTIR spectroscopy where characteristic
‒
1
bands appeared at 1723‒1699 cm were assigned to two carbonyl (C=O) functionalities of α,β-unsaturated

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1
ketone. H NMR spectroscopy revealed the synthesis of target compounds by the appearance of prominent
1
3
signals for unsaturated unit alongside disappearance of the methyl protons. C NMR spectroscopy further
confirmed the formation of 4a‒n by the appearance of aromatic carbons at their respective chemical shift
values.
Scheme 1. Synthesis of carbazole-chalcone hybrids (4a-n).
2
2
.2. Biology
.2.1. Urease inhibition
With the aim to explore a new class of compounds incorporating two distinct pharmacophores in one
combined unit, carbazole-chalcones (4a-n) were investigated for their biological potential against urease
enzyme. Thiourea was used as an internal standard (IC50 = 20.8 ± 0.59 µM). These medicinal chemistry
efforts have produced several potent inhibitors bearing various substituents. In vitro biological activity results
are summarized in Table 1.

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Table 1. In vitro urease inhibitory potential of carbazole-chalcone hybrids (4a–n)
Urease inhibition
Compound
Substituent (Ar)
%
Inhibition at 0.5 mM
IC50 ± SEM (µM)
1
2
9
9.3
4.5
4.8
—
4
4
4
a
b
c
—
8.93 ± 0.21
1
2
0.9
4.9
—
—
4
4
d
e
9
5.1
24.68 ± 0.94
4
f
1
9
8.6
4.4
—
4
4
g
14.15 ± 0.50
h
2
9.3
—
—
4
i
7
.9
4
j

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9
8.5
2.6
6.88 ± 0.42
149.02 ± 2.04
149.9 ± 1.02
4
k
2
4
l
7
7
4.5
8.7
4
m
24.78 ± 1.96
4
n
Thiourea
—
98.0
20.8 ± 0.59
2
.3. Molecular docking and structure-activity relationship analyses
Compounds 4k, 4c, 4f, 4n, 4h and 4l exhibited anti-urease activities in the range of 6.88‒149 µM. The mode
of interaction of these compounds was predicted by molecular docking in the active site of urease. The
binding interactions, docking scores and bond distances of active ligand with its interacting residues are
tabulated in Table 2. Among all the compounds, 4k and 4c were the most active compounds. Compound 4h
exhibited activity with an IC50 value of 14.15 ± 0.50 µM, whereas 4f and 4n showed comparable strength to
standard with IC50 values of ~24 µM. Compound 4l was found to be the least active compound (~149 µM). A
comparative structure-activity relationship of selected inhibitors with substitutional effect is represented in
Fig. 2.
The docked view of the most active compound 4k suggests that the compound is fitted neatly at the active
site interface and mediates interaction with both nickel atoms present in the active site. The propenone
oxygen facilitates bidentate interaction with the Ni1 and Ni2 at a distance of 2.35 and 2.51 Å, respectively.
The side chain (NE2) of His222 further donates H-bond to this oxygen. The carbazole moiety interacts with
the side chain of Cys322 and His323 and stabilized via hydrophobic interactions. The binding score of 4k is
higher than all the other compounds suggesting its highest activity. Similar to 4k, compound 4c also mediates

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metal-ligand interactions with both the nickel atoms and His222. Moreover, the bromophenyl ring in 4c
makes contact with the side chains of His139, Phe274 and His137, while its carbzole moiety forms
hydrophobic interactions with the side chain of His323 at the entrance of the active site. Furthermore, in
compound 4h, the carbonyl moiety interacts with the Ni2 at a distance of 2.25 Å while the substituted
nitrophenyl ring interacts with the main chain nitrogen of His275. Moreover, side chains of His137 and
Phe274 provide π‒π interactions. Compounds 4f and 4n exhibited IC50 in the range of 24 µM. Both
compounds interact with the nickel atoms while their long hydrocarbon chain remains surface exposed and
do not contribute in protein-ligand binding. The furan oxygen of 4n accepts hydrogen bond from the side
chain –OH of Thr301. Finally, the docked view of the least active compound 4l showed interaction with the
Ni1 and His222, while several residues (His137, His272, His323) provide π‒π interactions. The docking
interactions of each compound are tabulated in Table 2. The docking score shows strong correlation with our
experimental IC50 values, and a linear trend between experimental and docking results was observed. The
binding modes of all the active compounds are presented in Fig. 3.
Fig. 2. Structure-activity relationship of compound 4k with 4c, 4f, and 4h.

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Fig. 3. Docked view of compounds 4k, 4c, 4f, 4n, 4h and 4l in the active site of urease. Compounds are
presented in salmon sticks, active site residues are depicted in tan sticks, nickel atoms are shown in green ball
model, hydrogen bonds are displayed in black lines, metal-ligand interactions are shown in red dotted lines,
and the protein is depicted in gold ribbon.
Table 2. Docking scores and protein-ligand interactions of selected carbazole-chalcone hybrids.
Binding interactions
Compound
Docking Score
Ligand atoms
O28
Receptor atoms
NE2-His222
Ni1
Interaction type Distance (Å)
4
k
‒7.98
HBA
Metal
Metal
π‒π
HBA
Metal
Metal
π‒π
HBA
Metal
π‒π
π‒π
Metal
Metal
3.34
2.35
2.51
3.37
2.67
2.33
2.30
2.96
2.66
2.25
3.45
2.72
2.25
2.27
O28
O28
Ni2
6
-ring
5ring-His137
NE2-His222
Ni1
4
c
‒7.45
‒7.22
‒7.00
O28
O28
O28
Ni2
6
-ring
O65
O28
5ring-His137
N-His275
Ni2
6ring-Phe274
5ring-His137
Ni1
4
h
6
6
-ring
-ring
4
f
O28
O28
Ni2

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4
n
‒6.97
‒3.64
O28
Ni1
Metal
Metal
HBA
HBA
Metal
π‒π
2.44
2.45
1.99
2.93
2.33
3.62
3.46
3.23
O28
O35
O28
O28
Ni2
OG1-Thr301
NE2-His222
Ni1
4
l
5
6
6
-ring
-ring
-ring
5ring-His323
5ring-His137
5ring-His275
π‒π
π‒π
HBA: Hydrogen Bond Acceptor; HBD: Hydrogen Bond Donor.
3
. Conclusions
A series of hybrid compounds (carbazole-chalcones) containing a heterocyclic and non-heterocyclic motif
was accessed via a facile synthetic approach. In vitro biochemical assay showed that several compounds
inhibited the urease enzyme with significantly greater efficacy compared to standard drug (thiourea).
However, the preliminary structure-activity relationship analysis suggested the strong dependence of
inhibition strength on appropriately substituted aryl/heteroaryl ring of chalcone motif which may enforce to
generate an adaptable conformation in the active site of the enzyme. Importantly, compounds 4c and 4k
turned out to be the lead candidates with superior efficacy displaying IC50 values of 8.93 ± 0.21 and 6.88 ±
0
.42 µM, respectively. In addition, the in vitro inhibitory results produced by these chemical scaffolds were
rationalized using molecular docking tools. Hence, carbazole-chalcone hybrids can serve as a new combined
template for the development of novel urease inhibitors.
4
4
. Experimental
.1. General chemistry methods
Unless otherwise noted, all materials were obtained from commercial suppliers (Aldrich and Merck
companies) and used without further purification. Thin layer chromatography (TLC) was performed on
Merck DF-Alufoilien 60F254 0.2 mm precoated plates. Product spots were visualized under UV light at 254.
Melting points were recorded on a Stuart melting point apparatus (SMP3) and are uncorrected. Infra-red (IR)
1
spectra were recorded on FTS 3000 MX, Bio-Rad Merlin (Excalibur model) spectrophotometer. H NMR
spectra were recorded on a Bruker Avance (300 MHz) spectrometer. Chemical shifts () are quoted in parts
per million (ppm) downfield of tetramethylsilane, using residual solvent as internal standard (CDCl3 at 7.26

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and DMSO-d6 at 2.50 ppm). Abbreviations used in the description of resonances are: s (singlet), d (doublet), t
1
3
(
triplet), q, (quartet), m (multiplet), Ar (aromatic). Proton-decoupled C NMR spectra were recorded on a
Bruker Avance (75 MHz) spectrometer using deuterated solvent as internal standard (CDCl3 at 77.0 and
DMSO-d6 at 39.52 ppm). The elemental analysis was performed on Leco CHNS-932 Elemental Analyzer,
Leco Corporation (USA).
4
.2. General Procedure for the synthesis of N-octylcarbazole (2)
To a stirred mixture of carbazole (1.0 equiv) and octyl bromide (1.05 equiv) in benzene (25 mL) was added
0% KOH solution (60 mL) followed by tetrabutylammonium bromide (TBAB, 2.5 mol%) and mixture was
5
heated at 80 °C for 3 h. The reaction progress was monitored by thin layer chromatography (TLC). On
completion of reaction, the mixture was cooled to room temperature. The organic layer was separated and
diluted with water (50 mL), extracted with ethyl acetate (3 × 20 mL), dried (MgSO4), filtered and
concentrated in vacuo to afford the desired product [70]. The data was consistent to those reported in
literature [72].
–
1
1
Yield: 84%; Rf: 0.76 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2846 (Csp3–H), 1589, 1497 (C=C); H NMR
(300 MHz, CDCl3): δ 7.85–7.79 (m, 2H), 7.27–7.21 (m, 2H, ), 4.41 (t, 2H, J = 7.2 Hz), 1.73–1.51 (m, 12H),
1
3
0
3
.86 (t, 3H, J = 7.2 Hz); C NMR (75 MHz, CDCl3): δ 133.9, 125.2, 121.1, 119.8, 118.0, 114.1, 63.5, 39.4,
3.8, 30.4, 29.7, 27.1, 24.3; Anal. Calcd. for C20H25N: C, 85.97; H, 9.02; N, 5.01. Found: C, 85.86; H, 8.96;
N, 4.93.
.3. General procedure for the synthesis of 3,6-diacetyl-N-octylcarbazole (3)
To a stirred solution of N-octyl carbazole 2 (1.0 equiv) in chloroform was added a solution of acetyl chloride
2.0 equiv) in chloroform followed by aluminum chloride (1.0 equiv) at 0 °C. After complete addition, the
4
(
reaction mixture was stirred at room temperature for 4 h. The reaction mixture was diluted with water,
extracted with chloroform and concentrated under reduced pressure. The crude solid was recrystallized from
ethanol to afford the diacetylated carbazole 3 [71].
–
1
Yield: 92%; m.p.: 87–89 °C; Rf: 0.66 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2859 (Csp3–H), 1710
1
(
C=O); H NMR (300 MHz, DMSO-d6): δ 7.97–7.93 (m, 2H), 7.77–7.69 (m, 2H), 7.49–7.43 (m, 2H), 4.12

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1
3
(
t, 2H, J = 7.8 Hz), 2.79 (s, 3H), 1.78–1.63 (m, 12H), 0.79 (t, 3H, J = 7.2 Hz); C NMR (75 MHz, DMSO–
d6): δ 197.8, 136.4, 129.2, 125.8, 123.5, 121.0, 118.1, 65.2, 38.9, 37.6, 33.1, 31.4, 29.7, 27.5, 21.9; Anal.
Calcd. for C24H29NO2: C, 79.30; H, 8.04; N, 3.85. Found: C, 79.23; H, 7.96; N, 3.77.
4
.4. General procedure for the synthesis of substituted carbazole‒chalcones (4a-n)
To a stirred mixture of 3,6-diacetyl-N-octylcarbazole 3 (1.0 equiv) was added appropriate aldehyde (1.0
equiv) in methanol, followed by 6M NaOH. The reaction mixture was stirred at room temperature for 40 min
and then cooled to 0 °C. The precipitated solid was filtered, washed with cold water and methanol, dried and
recrystallized (MeOH) to afford the desired carbazole-chalcones (4a-n) in good isolated yields [70].
4
.4.1. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-phenylprop-2-en-1-one (4a)
–
1
Yield: 73%; m.p.: 140–142 °C; R : 0.56 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2849 (C –H), 1715
f
sp3
1
(
C=O), 1590, 1501 (C=C); H NMR (300 MHz, DMSO–d6): δ 8.04 (d, 1H, J = 7.8 Hz), 7.99 (d, 1H, J = 7.8
Hz), 7.89–7.77 (m, 3H), 7.59–7.49 (m, 2H), 7.39–7.33 (m, 3H), 7.27–7.19 (m, 3H), 4.11 (t, 2H, J = 7.8 Hz),
1
3
2
4
1
3
4
.50 (s, 3H), 1.79-1.55 (m, 12H), 0.89 (t, 3H, J = 7.2 Hz); C NMR (75 MHz, DMSO–d6): δ 198.8, 190.1,
5.9, 142.3, 136.2, 133.8, 131.9, 130.7, 129.3, 127.9, 126.6, 125.8, 123.5, 121.8, 120.5, 117.1, 115.4, 113.6,
11.9, 110.2, 59.4, 39.6, 33.4, 31.6, 30.3, 29.2, 28.0, 21.1; Anal. Calcd. for C31H33NO2: C, 82.45; H, 7.37; N,
.10. Found: C, 82.37; H, 7.28; N, 3.02.
.4.2. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(2-chlorophenyl)prop-2-en-1-one (4b)
–
1
Yield 69%; m.p.: 101–103 °C; Rf: 0.52 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2839 (Csp3–H), 1719
1
(
C=O), 1600, 1507 (C=C); H NMR (300 MHz, DMSO–d6): δ 8.09 (d, 1H, J = 7.8 Hz), 7.98 (d, 1H, J = 7.2
Hz), 7.85–7.79 (m, 3H), 7.60–7.49 (m, 2H), 7.43–7.35 (m, 2H), 7.19–7.10 (m, 3H), 4.19 (t, 2H, J = 7.2 Hz),
1
3
2
1
1
7
4
.65 (s, 3H), 1.71–1.53 (m, 12H), 0.78 (t, 3H, J = 7.2 Hz); C NMR (75 MHz, DMSO–d6): δ 196.9, 190.5,
45.0, 141.2, 135.2, 133.8, 131.0, 130.3, 129.3, 127.9, 125.9, 125.0, 123.5, 121.9, 120.0, 119.3, 117.8, 114.7,
13.5, 112.4, 111.6, 55.9, 39.6, 34.7, 33.0, 30.9, 29.5, 27.3, 26.1, 21.3; Anal. Calcd. for C31H32ClNO2: C,
6.60; H, 6.64; N, 2.88. Found: C, 76.51; H, 6.56; N, 2.79.
.4.3. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(2-bromophenyl)prop-2-en-1-one (4c)

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–
1
Yield: 67%; m.p.: 217–220 °C; Rf: 0.55 (20% EtOAc/n-hexane); FTIR (ATR, cm ) ) 2843 (Csp3–H), 1711
1
(
C=O), 1598, 1497 (C=C); H NMR (300 MHz, DMSO–d6): δ 8.14 (d, 1H, J = 7.8 Hz), 7.80 (d, 1H, J = 7.8
Hz), 7.77–7.73 (m, 3H), 7.57–7.51 (m, 2H), 7.39–7.31 (m, 2H), 7.21–7.11 (m, 3H), 4.07 (t, 2H, J = 7.3 Hz),
1
3
2
1
1
7
4
.45 (s, 3H), 1.72-1.60 (m, 12H), 0.87 (t, 3H, J = 7.1 Hz); C NMR (75 MHz, DMSO–d6): δ 197.6, 191.1,
45.6, 142.7, 135.7, 133.3, 131.3, 130.5, 129.8, 127.5, 126.9, 125.4, 122.8, 121.4, 120.9, 119.1, 117.4, 115.6,
13.9, 111.9, 110.4, 58.9, 38.6, 33.9, 32.6, 31.3, 29.9, 28.8, 27.0, 20.7; Anal. Calcd. for C31H32BrNO2: C,
0.19; H, 6.08; N, 2.64. Found: C, 70.10; H, 6.01; N, 2.56.
.4.4. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(3-bromophenyl)prop-2-en-1-one (4d)
–
1
Yield 63%; m.p.: 211–213 °C; Rf: 0.58 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2829 (Csp3–H), 1720
1
(
C=O), 1598, 1491 (C=C); H NMR (300 MHz, DMSO–d ): δ 8.19 (d, 1H, J = 7.8 Hz), 7.97 (d, 1H, J = 7.8
6
Hz), 7.83–7.75 (m, 3H), 7.67–7.53 (m, 2H), 7.43–7.37 (m, 2H), 7.19–7.07 (m, 3H), 4.24 (t, 2H, J = 7.6 Hz),
1
3
2
1
1
.35 (s, 3H), 1.47-1.32 (m, 12H), 0.93 (t, 3H, J = 7.2 Hz); C NMR (75 MHz, DMSO–d6): δ 197.0, 190.1,
45.4, 141.7, 135.3, 133.9, 130.8, 130.0, 129.3, 127.7, 126.4, 125.3, 123.7, 121.8, 119.5, 118.0, 116.6, 113.7,
11.6, 110.5, 109.9, 108.5, 58.2, 39.0, 33.4, 31.8, 28.3, 27.6, 23.6, 21.4, 15.6; Anal. Calcd. for C31H32BrNO2:
C, 70.19; H, 6.08; N, 2.64. Found: C, 70.11; H, 6.00; N, 2.54.
.4.5. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(4-bromophenyl)prop-2-en-1-one (4e)
4
–
1
Yield: 68%; m.p.: 201-203 °C; Rf: 0.54 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2832 (Csp3–H), 1716
1
(
C=O), 1592, 1497 (C=C); H NMR (300 MHz, DMSO–d6): δ 8.05 (d, 1H, J = 7.2 Hz), 7.75 (d, 1H, J = 7.8
Hz), 7.79–7.67 (m, 3H), 7.61–7.53 (m, 2H), 7.37–7.29 (m, 3H), 7.21–7.17 (m, 2H), 4.10 (t, 2H, J = 7.8 Hz),
1
3
2
1
1
7
4
.29 (s, 3H), 1.39-1.27 (m, 12H), 0.98 (t, 3H, J = 7.6 Hz); C NMR (75 MHz, DMSO–d6): δ 198.0, 190.6,
46.4, 142.7, 136.3, 133.7, 131.8, 130.5, 129.8, 128.7, 126.9, 125.7, 123.8, 121.5, 120.9, 119.3, 117.0, 114.7,
12.6, 109.4, 108.1, 57.6, 39.4, 33.9, 30.8, 29.2, 27.1, 25.6, 23.4, 17.6; Anal. Calcd. for C31H32BrNO2: C,
0.19; H, 6.08; N, 2.64. Found: C, 70.10; H, 6.01; N, 2.55.
.4.6. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(4-chlorophenyl)prop-2-en-1-one (4f)
–
1
Yield 62%; m.p.: 120–123 °C; Rf: 0.58 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2835 (Csp3–H), 1710
1
(
C=O), 1698, 1502 (C=C); H NMR (300 MHz, DMSO–d6): δ 8.07 (d, 1H, J = 7.8 Hz), 7.89 (d, 1H, J = 7.6

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Hz), 7.79–7.71 (m, 3H), 7.63–7.54 (m, 2H), 7.37–7.29 (m, 3H), 7.17–7.11 (m, 2H), 4.23 (t, 2H, J = 7.3 Hz),
1
3
2
1
1
7
4
.43 (s, 3H), 1.71–1.59 (m, 12H), 0.80 (t, 3H, J = 7.2 Hz); C NMR (75 MHz, DMSO–d6): δ 196.1, 190.3,
45.7, 141.4, 135.9, 133.2, 131.7, 130.8, 129.4, 128.5, 126.9, 125.4, 123.8, 121.7, 120.3, 119.0, 117.6, 114.7,
13.4, 110.6, 108.5, 55.2, 39.0, 35.7, 32.6, 30.8, 28.5, 26.4, 21.9, 16.2; Anal. Calcd. for C31H32ClNO2: C,
6.60; H, 6.64; N, 2.88. Found: C, 76.53; H, 6.54; N, 2.80.
.4.7. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(2-nitrophenyl)prop-2-en-1-one (4g)
–
1
Yield 66%; m.p.: 102–105 °C; Rf: 0.59 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2831 (Csp3–H), 1699
1
(
C=O), 1593, 1501 (C=C); H NMR (300 MHz, DMSO–d6): δ 8.25 (d, 1H, J = 8.1 Hz), 8.15 (d, 1H, J = 8.4
Hz), 8.00–7.88 (m, 3H), 7.63–7.61 (m, 3H), 7.55–7.44 (m, 2H), 7.28–7.17 (m, 2H), 4.36 (t, 2H, J = 7.3 Hz),
1
3
2
1
1
7
4
.75 (s, 3H), 1.42–1.32 (m, 12H), 0.92 (t, 3H, J = 7.1 Hz); C NMR (75 MHz, DMSO–d ): δ 197.7, 188.3,
6
44.1, 143.9, 140.9, 136.8, 134.3, 132.4, 130.9, 130.0, 129.8, 128.8, 127.4, 127.1, 124.5, 123.0, 122.8, 122.1,
09.4, 109.0, 68.1, 38.6, 31.7, 30.3, 29.3, 28.9, 27.2, 26.7, 23.7, 22.6, 14.1; Anal. Calcd. for C31H32ClNO2: C,
4.98; H, 6.50; N, 5.64. Found: C, 74.87; H, 6.41; N, 5.57.
.4.8. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(3-nitrophenyl)prop-2-en-1-one (4h)
–
1
Yield 58%; m.p.: 108–110 °C; Rf: 0.57 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2841 (Csp3–H), 1723
1
(
C=O), 1599, 1507 (C=C); H NMR (300 MHz, DMSO–d6): δ 8.29 (d, 1H, J = 8.3 Hz), 8.04 (d, 1H, J = 8.4
Hz), 7.97–7.85 (m, 3H), 7.61–7.56 (m, 3H), 7.52–7.45 (m, 2H), 7.22–7.19 (m, 2H), 4.23 (t, 2H, J = 7.2 Hz),
1
3
2
1
1
.71 (s, 3H), 1.39–1.24 (m, 12H), 0.88 (t, 3H, J = 7.2 Hz); C NMR (75 MHz, DMSO–d6): δ 197.8, 188.6,
48.6, 144.0, 143.1, 140.3, 136.3, 134.2, 132.1, 130.3, 129.4, 128.1, 127.7, 125.6, 124.7, 124.1, 123.6, 122.4,
21.7, 110.6, 109.9, 108.5, 67.5, 38.9, 31.4, 29.1, 28.2, 26.8, 23.2, 22.0, 17.5; Anal. Calcd. for C31H32N2O4:
C, 74.98; H, 6.50; N, 5.64. Found: C, 74.89; H, 6.40; N, 5.54.
.4.9. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(4-nitrophenyl)prop-2-en-1-one (4i)
4
–
1
Yield 64%; m.p.: 113–115 °C; Rf: 0.56 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2839 (Csp3–H), 1719
1
(
C=O), 1610, 1503 (C=C); H NMR (300 MHz, DMSO–d6): δ 8.17 (d, 2H, J = 8.4 Hz), 8.16–8.11 (m, 2H),
7
.89–7.83 (m, 3H), 7.53–7.48 (m, 3H), 7.45–7.28 (m, 2H), 4.35 (t, 2H, J = 7.2 Hz), 2.73 (s, 3H), 1.37–1.26
1
3
(
m, 12H), 0.91 (t, 3H, J = 7.5 Hz); C NMR (75 MHz, DMSO–d6): δ 196.8, 187.6, 147.3, 143.5, 142.7,

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1
6
39.3, 136.1, 134.9, 133.6, 131.3, 130.4, 129.6, 128.0, 126.7, 125.3, 123.7, 122.3, 121.5, 111.4, 109.6, 108.9,
7.9, 39.2, 30.7, 29.5, 28.6, 25.4, 23.9, 21.7, 17.4; Anal. Calcd. for C31H32N2O4: C, 74.98; H, 6.50; N, 5.64.
Found: C, 74.90; H, 6.41; N, 5.56.
.4.10. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(3-methoxyphenyl)prop-2-en-1-one (4j)
4
–
1
Yield 65%; m.p.: 212–215 °C; Rf: 0.49 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2821 (Csp3–H), 1713
1
(
C=O), 1596, 1489 (C=C); H NMR (300 MHz, DMSO–d6): δ 8.03 (d, 1H, J = 8.2 Hz), 7.81–7.73 (m, 3H),
7
1
1
1
7
4
.61–7.53 (m, 3H), 7.31–7.23 (m, 2H), 6.98–6.81 (m, 3H), 4.29 (t, 2H, J = 7.5 Hz), 3.35 (s, 3H), 2.56 (s, 3H),
1
3
.41–1.33 (m, 12H), 0.83 (t, 3H, J = 7.3 Hz); C NMR (75 MHz, DMSO–d6): δ 197.8, 188.7, 157.6, 146.2,
40.0, 136.9, 132.6, 130.9, 129.4, 127.9, 126.2, 123.8, 122.4, 121.7, 119.3, 115.6, 114.2, 113.5, 112.1, 111.5,
10.9, 108.2, 61.2, 56.9, 31.4, 30.8, 29.7, 28.4, 25.3, 22.2, 17.1; Anal. Calcd. for C H NO : C, 79.80; H,
3
2
35
3
.32; N, 2.91. Found: C, 79.71; H, 7.25; N, 2.83.
.4.11. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(4-methoxyphenyl)prop-2-en-1-one (4k)
–
1
Yield 61%; m.p.: 220–223 °C; Rf: 0.51 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2838 (Csp3–H), 1716
1
(
C=O), 1599, 1500 (C=C); H NMR (300 MHz, DMSO–d6): δ 7.98 (d, 1H, J = 8.5 Hz), 7.73–7.68 (m, 3H),
7
.57–7.51 (m, 3H), 7.27–7.19 (m, 3H), 6.83 (d, 2H, J = 7.6 Hz), 4.17 (t, 2H, J = 7.3 Hz), 3.56 (s, 3H), 2.34
1
3
(
s, 3H), 1.67–1.53 (m, 12H), 0.81 (t, 3H, J = 7.4 Hz); C NMR (75 MHz, DMSO–d6): δ 198.2, 187.2, 157.1,
1
1
45.7, 140.9, 136.4, 131.2, 130.0, 129.6, 128.3, 125.9, 123.5, 121.9, 121.0, 120.4, 118.5, 116.2, 113.7, 111.8,
10.5, 109.2, 63.0, 55.4, 30.6, 29.4, 28.0, 26.4, 25.7, 23.8, 21.5, 16.9; Anal. calcd. for C32H35NO3: C, 79.80;
H, 7.32; N, 2.91. Found: C, 79.70; H, 7.23; N, 2.81.
.4.12. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(4-N,N-dimethylphenyl)prop-2-en-1-one (4l)
4
–
1
Yield 59%; m.p.: 150–152 °C; Rf: 0.53 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2831 (Csp3–H), 1709
1
(
C=O), 1592, 1488 (C=C); H NMR (300 MHz, DMSO–d6): δ 7.93 (d, 1H, J = 8.1 Hz), 7.80–7.71 (m, 3H),
7
.61–7.54 (m, 3H), 7.21–7.13 (m, 3H), 6.79 (d, 2H, J = 7.3 Hz), 4.31 (t, 2H, J = 7.6 Hz), 2.98 (s, 6H), 2.28
1
3
(
s, 3H), 1.59–1.27 (m, 12H), 0.73 (t, 3H, J = 7.3 Hz); C NMR (75 MHz, DMSO–d6): δ 196.5, 186.7, 149.2,
1
45.0, 139.9, 133.6, 131.9, 129.4, 128.1, 127.8, 127.2, 125.9, 124.5, 123.8, 122.4, 121.7, 119.2, 117.6, 115.3,

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1
8
4
12.6, 110.9, 108.2, 61.6, 43.3, 31.6, 30.8, 29.7, 28.2, 25.4, 22.5, 19.1; Anal. Calcd. for C33H38N2O2: C,
0.13; H, 7.74; N, 5.66. Found: C, 80.02; H, 7.67; N, 5.57.
.4.13. 1-(6-Acetyl-9-octyl-9H-carbazole-3-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (4m)
–
1
Yield 68%; m.p.: 236–239 °C; Rf: 0.47 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2821 (Csp3–H), 1705
1
(
C=O), 1598, 1501 (C=C); H NMR (300 MHz, DMSO–d6): δ 8.08 (d, 1H, J = 8.4 Hz), 7.74–7.69 (m, 3H),
7
1
1
1
.65–7.56 (m, 2H), 7.27–7.21 (m, 2H), 6.68 (m, 2H), 4.16 (t, 2H, J = 7.4 Hz), 3.78 (s, 9H), 2.51 (s, 3H),
1
3
.73–1.40 (m, 12H), 0.79 (t, 3H, J = 7.2 Hz); C NMR (75 MHz, DMSO–d6): δ 196.7, 187.7, 154.2, 146.4,
41.3, 138.7, 132.8, 130.9, 129.7, 128.5, 127.7, 126.8, 125.3, 123.4, 122.0, 121.6, 118.5, 117.2, 116.7, 114.9,
12.8, 111.2, 109.7, 108.6, 60.2, 55.9, 31.2, 30.6, 29.9, 29.0, 27.6, 25.8, 23.5, 19.8; Anal. calcd. for
C H NO (541); C, 75.39; H, 7.26; N, 2.59. Found: C, 75.30; H, 7.17; N, 2.50.
3
4
35
5;
4
.4.14. 1-(6-Acetyl-9-octyl-9H-carbazol-3-yl)-3-(furan-2-yl)prop-2-en-1-one (4n)
–
1
Yield 57%; m.p.: 197–199 °C; Rf: 0.56 (20% EtOAc/n-hexane); FTIR (ATR, cm ) 2821 (Csp3–H), 1721
1
(
C=O), 1590, 1483 (C=C); H NMR (300 MHz, DMSO–d6): δ 7.93–7.80 (m, 2H), 7.71–7.63 (m, 3H), 7.53–
7
1
1
3
7
4
.47 (m, 2H), 7.23–7.11 (m, 2H), 6.89–6.73 (m, 2H), 4.20 (t, 2H, J = 7.2 Hz), 2.39 (s, 3H), 1.59–1.37 (m,
1
3
2H), 0.84 (t, 3H, J = 7.2 Hz); C NMR (75 MHz, DMSO–d6): δ 197.3, 188.1, 151.5, 145.7, 141.9, 139.7,
31.8, 130.5, 129.2, 128.7, 127.3, 126.1, 124.8, 123.7, 122.4, 121.2, 120.5, 117.6, 115.7, 113.5, 111.7, 59.2,
0.9, 29.6, 28.4, 25.4, 23.1, 20.7, 15.8; Anal. Calcd. for C29H31NO3: C, 78.88; H, 7.08; N, 3.17. Found: C,
8.79; H, 7.01; N, 3.09.
.5. Urease inhibition assay
The reaction mixtures consisting of 25 μL of Jack bean (Canavalia ensiformis) urease, 55 μL of buffer at pH
.8, 100 mM of urea, and 5 μL of various concentrations of test compounds (from 0.5 to 0.00625 mM) were
6
incubated at 30 °C for 15 min in 96-well plates. In kinetics experiments, various concentrations of both
substrates and test compounds were used. Subsequently 45 μL phenol reagents (1% w/v phenol and 0.005%
w/v sodium nitroprussside), and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% w/v NaOCl) were added
to each well. Urease activity through indophenols method was measured by the production of ammonia, as
described by Weatherburn [73]. After 50 min, the increasing absorbance at 630 nm was measured in a

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microplate reader (xMark™ Microplate Spectrophotometer, BIO-RAD). All reactions were performed in
triplicate in a final volume of 200 μL. Thiourea was used as the standard inhibitor of urease [74]. Finally, the
results were processed by software SoftMax Pro (Molecular Devices, CA, USA), MS-Excel and Ez-fit
programs. The percent inhibition was calculated from the formula given below:
%Inhibition = 100 ‒ (OD test /OD control) × 100
4
.6. Statistical analysis
The EZ-Fit Enzyme Kinetics program (Perrella Scientific Inc., Amherst, USA) was employed to calculate the
IC50 values.
4
.7. Molecular docking protocol
Molecular docking studies were carried out on the 3D-structure of urease enzyme (PDB code 4UBP,
resolution 1.55Å) using MOE docking suite (MOEv2014.09) [75]. Hydrogen atoms were added by
protonoate3D utility of MOE, and partial charges were calculated based on OPLSAA force field. Nickel (Ni)
parameters were set as mass = 58.6930, r = 1.4170Å, q = +2.0, and van der Waals well depth of 0.1225
2
kcal/mol. Protein atoms were minimized until gradient was reached to 0.1RMS kcal/mol/A . The structures
of compounds (4c, 4f, 4h, 4k, 4l, 4n) were prepared by MOE, hydrogen atoms were added and MMFF94x
force field was applied to calculate partial charges. Finally, compound’s structures were minimized until
2
gradient was reached to 0.1RMS kcal/mol/A . For docking, Alpha PMI docking algorithm and London dG
scoring function was applied. Induced fit docking method of MOE was applied. After docking thirty docked
conformations were saved for each ligand.
Disclosure statement
The authors declare that they have no conflict of interest.
Acknowledgement
Generous support from Higher Education Commission of Pakistan is gratefully acknowledged.
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Conflict of interest
The authors declare that they have no conflict of interest.
Research Highlights




New carbazole-chalcone hybrids were designed and synthesized.
Good yields and broad functional group tolerance were observed.
Compounds 4k and 4c were identified as the potent and lead molecules.
Molecular docking analysis was performed to delineate several key binding interactions.

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Graphical Abstract

Figure 1

Figure 2