Synthesis of 2-acylated and sulfonated 4-hydroxycoumarins: In vitro urease inhibition and molecular docking studies

Article abstract of DOI:10.1016/j.bioorg.2016.04.005

Sixteen 4-hydroxycoumarin derivatives were synthesized, characterized through EI-MS and ¹H NMR and screened for urease inhibitory potential. Three compounds exhibited better urease inhibition than the standard inhibitor thiourea (IC₅₀ = 21 ± 0.11 μM) while other four compounds exhibited good to moderate inhibition with IC₅₀ values between 29.45 ± 1.1 μM and 69.53 ± 0.9 μM. Structure activity relationship was established on the basis of molecular docking studies, which helped to predict the binding interactions of the most active compounds.

Full text of DOI:10.1016/j.bioorg.2016.04.005

Accepted Manuscript
Synthesis of 2-Acylated and Sulfonated 4-hydroxycoumarins: In vitro Urease
Inhibition and Molecular Docking Studies
Umer Rashid, Fazal Rahim, Muhammad Taha, Muhammad Arshad, Hayat
Ullah, Tariq Mahmood, Muhammad Ali
PII:
S0045-2068(16)30038-4
DOI:
http://dx.doi.org/10.1016/j.bioorg.2016.04.005
YBIOO 1903
Reference:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
29 February 2016
22 April 2016
25 April 2016
Please cite this article as: U. Rashid, F. Rahim, M. Taha, M. Arshad, H. Ullah, T. Mahmood, M. Ali, Synthesis of
2-Acylated and Sulfonated 4-hydroxycoumarins: In vitro Urease Inhibition and Molecular Docking Studies,
Bioorganic Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bioorg.2016.04.005
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Synthesis of 2-Acylated and Sulfonated 4-hydroxycoumarins: In vitro Urease Inhibition
and Molecular Docking Studies
Umer Rashid1†, Fazal Rahim2†, Muhammad Taha3,4, Muhammad Arshad5, Hayat Ullah2, Tariq
Mahmood1, Muhammad Ali1*
1Department of Chemistry, COMSATS Institute of Information Technology, University Road,
Abbottabad-22060, KPK, Pakistan
2Department of Chemistry, Hazara University, Mansehra, KPK, Pakistan
3Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),
Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia.
4Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia
5Institute of Chemistry, University of the Punjab, Quaid-i-Azam Campus, Lahore-54590,
Pakistan.
Abstract:
Sixteen 4-hydroxycoumarins derivatives were synthesized, characterized through EI-MS and
1HNMR and screened for urease inhibitory potential. Three compounds exhibited better urease
inhibition than the standard inhibitor thiourea (IC50= 21 ± 0.11 μM and other four compounds
exhibited good to moderate inhibition with IC50 values between 29.45 ± 1.1 μM and 69.53 ± 0.9
μM. Structure activity relationship was established on the basis of molecular docking studies,
which helped to predict the binding interactions of the most active compounds.
Keywords: Synthesis, 2-Acylated and Sulfonated 4-hydroxycoumarin, Urease Inhibition,
Molecular Docking, SAR.
____________________________________________________
*Corresponding author: muhammad_ali@ciit.net.pk
Ph. # +92 992 383 591-4; Ext. 314
†Both authors have contributed equally to this work.
1

1.0. Introduction
Urease (urea amidohydrolase, E.C.3.5.1.5) is a nickel containing metallo-enzyme found in
plants, bacteria, fungi and soil. Ureases derived from different sources have different structural
features; however, they share more than 50% amino acids characteristics. Urease catalyzes the
hydrolysis of urea into ammonia and carbamate, the later produces ammonia and carbon dioxide
on further decomposition [1, 2]. Urease is involved in human and animal pathogenicity of
hepatic encephalopathy, hepatic coma urolithiasis, pyelonephritis, gastric and peptic ulcers;
while urinary catheter encrustation are also caused by ammonia produced by ureases [3-4].
Urease inhibitors can be considered as tool to control the damaging effects of ureolytic bacterial
infections in humans, which occur commonly in the developed countries [5-6].
Ureases are inhibited by different classes of compounds. Urease inhibitors can be broadly
classified into two categories; organic compounds and organometallics [7]. The former category
includes hydroxamic acid and its derivatives [8], triazoles, coumarins [9], isatins,
semicarbazones [10], Schiff bases [11], urea derivatives [12], oxadiazoles [13], piperidines [14];
however, the later includes organophosphorous/phosphinic inhibitors [15]. Coumarin (1,2-
benzopyrone) derivatives constitute one of the most common families of green plant secondary
metabolites. Several type of coumarins have been reported to display multiple biological
properties, e.g. [16-17]. Many products which contain a coumarin subunit exhibit biological
activities, such as molluscicidal, anthelmintic, hypnotic, and insecticidal activities [18]. The
medicinal properties of coumarins include the inhibition of platelet aggregation, cytochrome
P450 and steroid 5α-reductase [19].
4-Hydroxycoumarin is an interesting compound found in many plants and particularly known as
the natural precursor of dicoumarol, a powerful anticoagulant that acts as a vitamin K antagonist
[20]. In addition, recent studies in a collaborative effort between chemists and biologists have
shown that a number of 4-hydroxycoumarins show biological activity as potent non-nucleoside
RT inhibitors [21]; HIV integrase [22] or HIV protease [23] inhibitors, promising characteristics
have been identified. Khan et al reported a variety of bis-coumarins as urease inhibitors with IC50
values ranging from m 15.06–91.35 µM [24]. Only compound 1 (Figure 1a, R = H) showed
good urease inhibition. They concluded that the two hydroxyl groups present on two lactone
rings of bis-coumarin molecule may be responsible for inhibitory activity while the decline in
activity was rationalized due to the steric hindrance of bulky groups (R). In the current study, we
2

are reporting the synthesis of simple and new derivatives of 4-hydroxycoumarins (Figure 1b)
with better urease inhibitory potentials than the reported bis-coumarins. The molecular docking
studies were performed to determine the binding interactions of these compounds.
a)
b)
R
OH
OH
O
OH
R
O
O
O
O
(1)
Current Work
.
Figure 1: (a) Structure of the previously identified bis-coumarin (1) as urease inhibitor and; (b)
the structural framework of the current study.
2.0. Results and Discussion
2.1. Chemistry
A mixture of acyl chloride (1 mmol) and 4-hydroxycoumarin (1 mmol) was dissolved in dry
pyridine (5 mL), and stirred at room temperature for 10 minutes. Water was added to precipitate
out the product, which was filtered on suction and washed with excess of water followed by
EtOH. The products were finally recrystallized to obtain pure compounds (1-16).
Scheme-1: Synthesis of coumarin analogs (1-16)
3

Table-1: Synthesis of various derivatives of coumarins (1-16)
Yield
Yield
(%)
S. No.
R
S. No.
R
(%)
-CO-CH3
90
73
83
82
88
80
81
74
74
1
9
-CO-CHCl2
-CO-C(CH3)3
70
78
84
86
89
85
82
2
3
4
5
6
7
8
10
11
12
13
14
15
16
-CO-(CH2)7 CH3
-CO-(CH2)8 CH3
-CO-(CH2)10 CH3
4

2.2 Urease Inhibitory Activity
Compounds 1-16 were screened for urease inhibitory potential. Some compounds among the
series showed potent urease inhibitory potential (Table-2). Compounds 5, 6, and 9 exhibited
excellent urease inhibition with IC50 values 13.01 ± 0.8, 17.09 ± 1.1, and 11.30 ± 1.7 μM,
respectively, which is better than the standard inhibitor thiourea (IC50 = 21 ± 0.11 μM).
Compound 4, 11, 14 and 16 exhibited good to moderate potential for urease inhibition with IC50
values of 30.87 ± 1.3, 29.45 ± 1.1, 56.2 ± 1.2 and 69.53 ± 0.9 μM, respectively.
Structure-activity relationship predicted that the urease inhibitory potential of molecules was
basically governed by the substitution pattern on main skeleton. It is obvious from Table 2, with
a few exceptions, that incorporation of alkyl chains was well tolerated. The 3, 4, 5-trimethoxy
phenyl substituted analog 9 (IC50 = 11.30 ±1.7 µM) displayed good potency even better than the
standard drug thiourea. At the same time, fusion of another phenyl ring (naphthalene) in
compound 10 furnished a substantial loss in the inhibitory potential. 1-decanone substituted
analog 5 (IC50 = 13.01 ± 0.8 M) and 1-dodecanone substituted analog 6 (IC50 = 17.09 ± 1.1
µM) exhibited outstanding inhibitory potential among the whole series. The greater potential of
the most active analog 9 might be due to having electron donating groups on the phenyl ring.
Similarly analogs 5 and 6 contain alkyl moieties, which are weakly, electron donor might be
responsible for these greater potentials. If we compare these more active compounds with
compounds 1, 2 and 3 which also contain alkyl moiety but show no inhibitions. The reason
behind this seems to be the nature of the alkyl group. We observed here that by increasing the
length of the alkyl chain, inhibitory potential is increased. This was confirmed from compound 4
which contains nonane-1-one moiety and show weaker potential for the inhibition of urease as
compared to the compounds 5 and 6, both of which contain longer alkyl groups. In case of the
sulfonated analogs, the methanesulfonyl substituted analog 11, 3-nitrophenyl sulfonyl analog 14
and naphthyl sulfonyl analog 16, having IC50 values 29.45±1.1, 56.2±1.2 and 69.53±0.9 μM,
respectively, showed good potential for urease inhibition. It is concluded that the replacement of
phenyl to naphthyl group, i.e. introduction of additional ring resulted in the substantial decrease
or loss in activity and this is the reason of the higher inhibitory potential of simple coumarins
when compared with the previously studied bis-coumarins by Khan et al. Our results are further
5

supported by molecular docking studies and the mean binding energies (Figure S-1, Supporting
Information).
Table-2: Urease inhibitory potential of compounds 1-16
S. No
IC50 ± SEMa(µ/M)
S. No
IC50 ± SEMa(µ/M)
11.30±1.7
NA
NA
NA
1
2
3
4
5
6
7
8
9
10
NA
29.45±1.1
NA
11
30.87 ± 1.3
13.01±0.8
17.09±1.1
NA
12
NA
13
56.2±1.2
NA
14
15
69.53±0.9
21 ± 0.11
NA
16
Thioureab
SEMa is the standard error of the mean, Thioureab standard inhibitor for anti-urease activity
NA stands for “Not Active”
2.3. Molecular Docking Studies
To investigate the plausible orientation of the synthesized compounds and the compound-
enzyme interactions, molecular docking studies were performed by using crystal structure of
Bacillus pasteurii (BP) in complex with acetohydroxamic acid (HAE, PDB ID 4UBP).
AutoDock 4.2 and a Lamarckian genetic algorithm (LGA) were used for protein-fixed ligand-
flexible docking calculations. In order to check the reliability of the docking method,
acetohydroxamic acid was docked into the active site of 4UBP. The root mean square deviation
(RMSD) between co-crystallized and re-docked conformation was found 0.981 Å suggesting
high docking reliability.
The active site of urease is located within the cavity or the crevice in its internal territory in
which HAE molecule chelates with two nickel ions (Ni798 and Ni799) via hydroxyl oxygen. The
key amino acid residues in the catalytic site of BPU are Ala170, His137, His139, Lys220,
His249, His275, Gly280, Cys322, His323, His324, Arg339, Ala363 and Asp363. His137, His139
and KCX220 residues are liganded to Ni799. Whereas, His249, His275, Asp363 residues are
6

liganded to Ni798. Carbamylated Lys490 (KCX220, a non-standard residue), acts as bridging
residue between two Ni ions.
We analyzed the computer generated molecular model of compounds and our analysis identified
that all active compounds interact well with Ni ions of the urease enzyme. The scoring functions,
i.e., the distance of interaction, coordination pattern, nature of interaction and the residues
involved in the interaction were carefully examined. The coordination pattern of the most active
compound 9 (IC50 = 11.30±1.7 µM) is shown in Figure 2. From the structure of the docking
conformation, it appeared that the greater activity of compound 9 is due to the formation of a
stable complex of two nickel atoms with oxygen of the methoxy group at a binding distance of
2.9 Å and HBA with Arg339 at a distance of 2.87 Å. The observed negative fitness values of
binding interactions (mean binding energy=12.08 kcal/mol) revealed that compound 9 is tightly
fitted into the active site of 4UBP.
Figure 2: Modeled mode of binding of most active compound 9 (Magenta) into the active site of
urease from Bacillus pasteurii (A) Ribbon form (B) Docked conformation showing interactions
with Ni bi-center (C) Surface form
The interaction pattern of compounds with moderate and weak activity (Compound 11 and
compound 16) revealed that Ni metal ligation in the ligand-enzyme complex is essential for
stabilizing a ligand-enzyme complex. Figure 3A revealed that compound 11 (IC50=29.45±1.1)
having methanesulfonyl group formed strong ligation with Ni798 at a binding distance of 1.0 Å.
While, Ni799 displaced away from sulfonyl oxygen at the distance of 3.0 Å and this looks to be
the apparent reason for the moderate in vitro activity of compound 11. Similarly sulfonyl oxygen
7

is pointed towards His249 and His275 and acts as a hydrogen bond acceptor. Taking into the
consideration of the fitness value, compound 11 have shown mean binding energy= 11.17
kcal/mol. By replacing methanesulfonyl group (compound 11) to 1-naphthosulfonyl (compound
16) the urease inhibition activity decreased (IC50=69.53±0.9). In Figure 3B, it can be seen that
although the compound 16 displaced away from nickel bi-center at the distance of 5.8 Å and 8.0
Å and show no metal ligation,however, it formed hydrogen bond with Asp224 and Arg339.
Mean binding energy of 8.29 kcal/mol is the possible explanation for the poor in vitro activity
of compound 16.
Figure 3: Docked conformation of compounds with moderately and weak urease inhibition
activity showing interactions with Ni bi-center and different amino acid residues (A) Compound
11 (Green) (B) Compound 16 (Orange red).
2.4. Urease Inhibition Assay
Reaction mixtures comprising one unit of urease enzyme (Bacillus pasteurii) solution and 55 μL
of buffers containing 100 mMol urea were incubated with 5 L of test compounds (1 mMol
°
concentration) at 30 C for 15 min in 96-well plates. Urease activity was determined by
measuring ammonia production using the indophenol’s method [25]. Momentarily, 45 L each of
phenol reagent and 70 L of alkali reagent were added to each well. The increasing absorbance
at 630 nm was measured after 50 min, using a micro-plate reader (Molecular Devices, USA). All
reactions were performed in triplicate in a final volume of 200 L. The results (change in
8

absorbance per min.) were processed by using the Soft-Max Pro s4.5.Software (Molecular
Devices, USA).
3.0. Conclusion
Synthesis and urease inhibition bioassay of new acylated and sulfonylated 4-hydroxycoumarins
has been carried out. Among the newly synthesized derivatives (1-16), compound 5, 6 and 9
were found to be highly potent for urease inhibition. The greater inhibitory potential of these
analogs as compared to the standard thiourea was also explained by performing in silico
molecular docking studies. This work allowed us to obtain a preliminary biological profile of the
series indicating which type of compounds might be suitable for further investigation and in
which direction the further optimization efforts should be pursued in future.
4.0. Experimental
4.1. General
NMR experiments were performed on Avance Bruker AM 400 MHz machine. CHN Analysis
was performed on a Carlo Erba Strumentazion-Mod-1106, Italy. Ultraviolet (UV) spectra were
recorded on Perkin–Elmer Lambda-5 UV/vis spectrophotometer in MeOH. Infrared (IR) spectra
were recorded on JASCO IR-A-302 spectrometer as KBr (disc). Electron impact mass spectra
(EI-MS) were recorded on a Finnigan MAT-311A (Germany) mass spectrometer. Thin layer
chromatography (TLC) was performed on pre-coated silica gel aluminum plates (Kieselgel 60,
254, E. Merck, Germany). Chromatograms were visualized by UV at 254 and 365 nm.
4.2. Docking Studies
Docking studies were carried out using Autodock 4.2 [26]. Gasteiger charges were added to the
ligand and maximum 6 number of active torsions are given to the lead compound using
AutoDock Tool. 2D structures of ligands converted to 3D in pdbqt format by Openbabel (ver.
2.3.1) [27]. Kollaman charges and solvation term were added to the protein structure. The Grid
for docking calculation was centered to cover the protein binding site residues and accommodate
ligand to move freely. Docking parameters were as follows: 30 docking trials, population size of
300, maximum number of energy evaluation ranges of 250000, maximum number of
generationsis 27,000, mutation rate of 0.02, cross-over rate of 0.8, Other docking parameters
were set to the software’s default values. The view of the docking results and analysis of their
surface with graphical representations were done using UCSF Chimera package [28].
9

4.3. General procedure for the synthesis of compounds (1-16)
A mixture of acyl chloride (1 mmol) and 4-hydroxycoumarin (1 mmol) dissolved in dry pyridine
(5 mL), was stirred at room temperature for 10 minutes, then water was added till precipitates
formed, filtered on suction and washed with excess of water followed by EtOH then
recrystallized to obtained pure products.
3-Acetyl-(4-hydroxycoumarin) (1)
1
Yield: 0.56 g (90%); Rf = 0.5 (Ethyl acetate: Hexane, 9:1); H-NMR (400 MHz, CDCl3):  7.86
(d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J = 7.8Hz, H-8),
7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6), 1.44 (s, 3H, -CH3); IR (KBr, cm–1) max 1553, 1610, 1726,
2928, 3426; UV/Vis. (CHCl3) λmax (log ε) 214.2 (4.35) nm; EI-MS (m/z) 204, (M+, 71), 189 (47),
162 (28), 120 (100), 92 (34), 77 (23), 63 (33), 51 (34); Anal. calcd. for C11H8O4 C 64.71; H 3.95;
Found: C 64.78; H 3.90.
3-Dichloroacetyl-(4-hydroxycoumarin) (2)
Yield: 0.35 g (70%); Rf = 0.64 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  8.41
(s, 1H, -CH), 7.86 (d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J
= 7.8Hz, H-8), 7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6); IR (KBr) max 750, 1560, 1640, 1710,
2970, 3410 cm–1; UV (CHCl3) λmax (log ε) 214.2 (4.42) nm; EI-MS (m/z) 442, (M+, 71), 189
(47), 162 (28), 120 (100), 92 (34), 77 (23), 63 (33), 51 (34); Anal. calcd for C11H6O4Cl2 C 48.38;
H 2.21; Found: C 48.46; H 2.25.
3-Pivaloyl-(4-hydroxycoumarin) (3)
Yield: 0.41 g (78%); Rf = 0.62 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  7.86
(d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J = 7.8Hz, H-8),
7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6), 1.48 (s, 9H, -CH3); IR (KBr) max 1550, 1618, 1730,
3010, 3390 cm–1; UV (CHCl3) λmax (log ε) 215.4 (4.71) nm; EI-MS (m/z) 442, (M+, 76), 189
(47), 162 (28), 120 (100), 92 (34), 77 (23), 63 (33), 51 (34); Anal. calcd for C14H14O4C 68.28; H
5.73; Found: C 68.25; H 5.50.
3-Nonanoyl-(4-hydroxycoumarin) (4).
10

Yield: 0.73 g (84%); Rf = 0.58 (Ethyl acetate: Hexane, 9:1); H1-NMR (500 MHz, CDCl3):  7.89
(d, 1H, J = 7.8 Hz, H-5), 7.55 (td, 1H, J = 7.55, J = 2.0 Hz, H-7), 7.30 (dd, 2H, J = 7.8 Hz, H-8),
7.23 (td, 2H, J = 7.8, J = 2.0 Hz, H-6), 1.59 (2H, t, -CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3),
1.14-1.64 (12H, m, -CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 0.84 (t, 3H, J = 6.6 Hz, -CH2-
CH2-CH2-CH2-CH2-CH2-CH2-CH3); IR (KBr) max 1558, 1612, 1730, 2940, 3610 cm–1; UV
(CHCl3) λmax (log ε) 214.7 (4.81) nm; EI-MS (m/z) 302, (M+, 53), 189 (34), 162 (58), 120 (100),
92 (67), 77 (6), 63 (27), 51 (18); Anal. calcd for C18H22O4 C 71.50; H 7.33; Found: C 71.60; H
7.5.
3-Decanoyl-(4-hydroxycoumarin) (5).
1
Yield: 0.86 g (86%); Rf = 0.47 (Ethyl acetate: Hexane, 9:1); H-NMR (400 MHz, CDCl3 7.86
(d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J = 7.8Hz, H-8),
7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6), 1.67 (2H, t, -CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3),
1.15-1.51 (14H, m, -CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 0.76 (t, 3H, J = 6.6 Hz, CH2-
CH2-CH2-CH2-CH2-CH2-CH2-CH3); IR (KBr) max 1559, 1608, 1709, 2925, 3398 cm–1; UV
(CHCl3) λmax (log ε) 212.8 (4.74) nm; EI-MS (m/z) 316, (M+, 55), 204 (14), 189 (40), 162 (58),
120 (100), 92 (56), 77 (6), 63 (25), 51 (15); Anal. calcd for C19H24O4 C 72.13; H 7.65; Found: C
72.20; H 7.70.
3- Lauroyl-(4-hydroxycoumarin) (6)
Yield: 0.46 g (89%); Rf = 0.54 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  7.86
(d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J = 7.8Hz, H-8),
7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6), 1.74 (2H, m, -CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3),
1.15-1.60 (14H, m, -CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3), 0.74 (t, 3H, J = 6.6 Hz, -
CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH3); IR (KBr) max 1544, 1614, 1705, 2940, 3410 cm–1; UV
(CHCl3) λmax (log ε) 209.8 (4.98) nm; EI-MS (m/z) 344, (M+, 70), 204 (5), 189 (3), 162 (71), 120
(100), 92 (37), 77 (5), 63 (15), 51 (33); Anal. calcd for C21H28O4 C 73.23; H 8.19; Found: C
73.30; H 8.20.
3-Benzoyl-(4-hydroxycoumarin) (7)
Yield: 0.70 g (85%); Rf = 0.62 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  7.86
(d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J = 7.8Hz, H-8),
7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6), 7.01 (d, 2H, J = 8.3 Hz, H-2/6), 6.70 (d, 2H, J = 8.3 Hz,
H-3/5), 5.89 (td, 1H, J = 7.8, J = 2.0 Hz, H-4); IR (KBr) max 1567, 1605, 1637, 1732, 2970,
11

3419 cm–1; UV (CHCl3) λmax (log ε) 201.6 (5.21) nm; EI-MS (m/z) 266, (M+, 15), 162 (4), 120
(8), 105 (100) 92 (5), 77 (44), 63 (3) 51 (15);
Anal. calcd for C16H10O4 C 72.18; H 3.79; Found: C 72.20; H 43.72.
3-(4-Methylbenzoyl-(4-hydroxycoumarin) (8)
Yield: 0.61 g (82%); Rf = 0.63 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  7.86
(d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J = 7.8Hz, H-8),
7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6), 7.12 (d, 2H, J = 8.3 Hz, H-2/6), 7.12 (d, 2H, J = 8.3 Hz,
H-3/5), 2.66 (s, 3H, -CH3); IR (KBr) max 1552, 1615, 1730, 2935, 3389 cm–1; UV (CHCl3) λmax
(log ε) 202.7 (5.12) nm; EI-MS (m/z) 442, (M+, 76), 321 (12), 281 (21), 265 (18), 249 (100), 162
(64), 120 (99), 92 (97), 63 (61); Anal. calcd for C17H12O4 C 72.85; H 4.32; Found: C 72.83; H
4.28.
3-(3, 4, 5-Trimethoxybenzoyl-(4-hydroxycoumarin) (9)
Yield: 0.80 g (73%); Rf = 0.61 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  7.86
(d, 2H, J = 7.8 Hz, H-5/5΄), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7/7΄), 7.32 (d, 2H, J = 7.8Hz, H-
8/8΄), 7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6/6΄), 7.01 (d, 2H, J = 8.3 Hz, H-2/6), 3.78 (s, 3H,
OCH3), 3.89 (s, 6H, -OCH3); IR (KBr) max 1594, 1629, 1726, 1750, 2930, 3425 cm–1; UV
(CHCl3) λmax (log ε) 201.9 (5.25) nm; EI-MS (m/z) 356, (M+, 30), 195 (100), 167 (7), 120 (3),
109 (9) 92 (3), 63 (2); Anal. calcd for C19H16O7 C 64.04; H 4.53; Found: C 64.10; H 4.58.
3-(1-Nephthoyl-(4-hydroxycoumarin) (10)
Yield: 0.82 g (83%); Rf = 0.48 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  8.25
(d,1H, J = 7.8 Hz, H-4), 8.25 (d,1H, J = 7.8 Hz, H-4), 7.86 (d, 2H, J = 7.8 Hz, H-2/8΄), 7.57
(td, 2H, J = 7.8, J = 1.9 Hz, H-7/7΄), 7.29-7.31 (m, 3H, H-3/5΄-7), 7.32 (d, 2H, J = 7.8 Hz, H-
8), 7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6); IR (KBr) max 1555, 1608, 1725, 2915, 3425 cm–1; UV
(CHCl3) λmax (log ε) 202.4 (5.20) nm; EI-MS (m/z) 442, (M+, 76), 321 (12), 281 (21), 265 (18),
249 (100), 162 (64), 120 (99), 92 (97), 63 (61); Anal. calcd for C20H12O4 C 75.94; H 3.82;
Found: C 75.88; H 3.90.
3-(Methanesulfonoyl-(4-hydroxycoumarin) (11)
Yield: 0.61 g (82%); Rf = 0.63 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  7.86
(d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J = 7.8Hz, H-8/8΄),
7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6), 3.46 (s, 3H, -CH3); IR (KBr) max 1050, 1550, 1614,
12

1720, 2923, 3468 cm–1; UV (CHCl3) λmax (log ε) 206.2 (4.87) nm; EI-MS (m/z) 442, (M+, 76),
321 (12), 281 (21), 265 (18), 249 (100), 162 (64), 120 (99), 92 (97), 63 (61); Anal. calcd for
C10H8O5S C 50.0; H 3.36; Found: C 50.03; H 3.39.
3-(Ethanesulfonoyl-(4-hydroxycoumarin) (12)
Yield: 0.49 g (88%); Rf = 0.56 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  7.86
(d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J = 7.8Hz, H-8),
7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6), 7.03 (s, 2H, H-3/5), 2.66 (s, 6H,CH3), 2.33 (s, 3H, -
CH3);IR (KBr) max 1046, 1550, 1610, 1729, 2938, 3460 cm–1; UV (CHCl3) λmax (log ε) 207
(4.84) nm; EI-MS (m/z) 442, (M+, 76), 321 (12), 281 (21), 265 (18), 249 (100), 162 (64), 120
(99), 92 (97), 63 (61); Anal. calcd for C11H10O5S C 51.96; H 3.96; Found: C 51.94; H 3.99.
3-(Mesitylenebenzosulfonoyl-(4-hydroxycoumarin) (13)
Yield: 0.74 g (80%); Rf = 0.68 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  7.86
(d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J = 7.8 Hz, H-8),
7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6), 7.03 (s, 2H, J = 7.8 Hz, H-3/5), 2.66 (s, 6H, J = 8.3 Hz,
CH3), 2.33 (s, 3H, -CH3); IR (KBr) max 1064, 1568, 1605, 1736, 2946, 3446 cm–1; UV (CHCl3)
λmax (log ε) 205.6 (4.80) nm; EI-MS (m/z) 442, (M+, 76), 321 (12), 281 (21), 265 (18), 249 (100),
162 (64), 120 (99), 92 (97), 63 (61); Anal. calcd for C18H16O5 C 62.78; H 4.68; Found: C 62.79;
H 4.65.
3-(3-Nitrobenzosulfonoyl-(4-hydroxycoumarin) (14)
Yield: 0.86 g (81%); Rf = 0.58 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  8.70
(s, 1H, H-2), 8.45 (d, 2H, J = 7.8 Hz, H- ), 8.37 (d, 1H, J = 7.8 Hz, H- ), 8.24 (td, 2H, J = 7.8, J
= 1.9 Hz, H- ),7.86 (d, 2H, J = 7.8 Hz, H-5), 7.57 (td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H,
J = 7.8Hz, H-8), 7.86 (d, 2H, J = 7.8 Hz, H-5);IR (KBr) max 1045, 1349, 1545, 1550, 1610,
1730, 2940, 3428 cm–1; UV (CHCl3) λmax (log ε) 205 (4.90) nm; EI-MS (m/z) 442, (M+, 76), 321
(12), 281 (21), 265 (18), 249 (100), 162 (64), 120 (99), 92 (97), 63 (61); Anal. calcd for
C15H9NO7S C 51.87; H 2.61; Found: C 51.90; H 2.60.
3-(4-Nitrobenzosulfonoyl-(4-hydroxycoumarin) (15)
Yield: 0.80 g (74%); Rf = 0.56 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  8.45
(d, 2H, J = 7.8 Hz, H-2/6), 8.37 (d, 1H, J = 7.8 Hz, H-3/5 ), 7.86 (d, 2H, J = 7.8 Hz, H-5), 7.57
(td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.32 (d, 2H, J = 7.8Hz, H-8), 7.27 (td, 2H, J = 7.8, J = 2.0 Hz,
13

H-6); IR (KBr) max 1061, 1348, 1544, 1548, 1612, 1732, 2930, 3448 cm–1; UV (CHCl3) λmax
(log ε) 205.6 (4.94) nm; EI-MS (m/z) 442, (M+, 76), 321 (12), 281 (21), 265 (18), 249 (100), 162
(64), 120 (99), 92 (97), 63 (61); Anal. calcd for C15H9NO7S C 51.87; H 2.61; Found: C 51.85; H
2.65.
3-(1-Nephthosulfonoyl-(4-hydroxycoumarin) (16)
Yield: 0.80 g (74%); Rf = 0.56 (Ethyl acetate: Hexane, 9:1); 1H-NMR (400 MHz, CDCl3):  8.27
(d, 1H, J = 7.8 Hz, H-4΄), 7.92 (d, 2H, J = 7.8 Hz, H-2/8΄), 7.86 (d, 2H, J = 7.8 Hz, H-5), 7.57
(td, 2H, J = 7.8, J = 1.9 Hz, H-7), 7.43-7.55 (m, 3H, H-3/5΄-7), 7.32 (d, 2H, J = 7.8 Hz, H-8),
7.27 (td, 2H, J = 7.8, J = 2.0 Hz, H-6); IR (KBr) max 1025, 1555, 1602, 1748, 2974, 3375 cm–1;
UV (CHCl3) λmax (log ε) 204.9 (4.96) nm; EI-MS (m/z) 442, (M+, 76), 321 (12), 281 (21), 265
(18), 249 (100), 162 (64), 120 (99), 92 (97), 63 (61); Anal. calcd for C19H12O5S C 64.76; H 3.43;
Found: C 64.78; H 3.40.
Acknowledgements: The author (Dr. M.A.) is grateful to Higher Education Commission of
Pakistan for providing a research grant under National Research Program for Universities vide
No. 20-1933/NRPU/R&D/HEC/12/5017.
14

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16

Graphical Abstract
Synthesis of 2-Acylated and Sulfonated 4-hydroxycoumarins:
In vitro Urease Inhibition and Molecular Docking Studies
Umer Rashid1†, Fazal Rahim2†, Muhammad Taha3,4, Muhammad Arshad5,
Hayat Ullah2, Tariq Mahmood1, Muhammad Ali1*
1Department of Chemistry, COMSATS Institute of Information Technology, University Road, Abbottabad-22060, KPK, Pakistan
2Department of Chemistry, Hazara University, Mansehra, KPK, Pakistan
3Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM),
Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia.
4Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia
5Institute of Chemistry, University of the Punjab, Quaid-i-Azam Campus, Lahore-54590, Pakistan.
Synthesis of coumarin analogs (1-16)
Modeled mode of binding of most active compound 9 (Magenta) into the active site of urease from Bacillus pasteurii (A) Ribbon
form (B) Docked conformation showing interactions with Ni bi-center (C) Surface form

Highlights





Acyl and Sulfonated coumarin derivatives are synthesized
Compounds are evaluated for their urease inhibitory potential
SAR is established on the basis of Molecular Modeling studies
A comparison with previously synthesized biscoumarins is also provided
New coumarin derivatives proved better inhibitors of urease as compared to previous
biscoumarin derivatives
17