The synthesis, structural elucidation and antimicrobial activity of 2- and 4-substituted-coumarinyl chalcones

Full text of DOI:10.1002/mrc.4414

Letter - spectral assignment
Received: 29 October 2015
Revised: 3 January 2016
Accepted: 7 January 2016
Published online in Wiley Online Library: 11 February 2016
(wileyonlinelibrary.com) DOI 10.1002/mrc.4414
The synthesis, structural elucidation and
antimicrobial activity of 2- and
4-substituted-coumarinyl chalcones
Thrineshen Moodley,a Mehbub Momin,a Chunderika Mocktar,b
Christina Kannigadua and Neil A. Koorbanallya*
regard to antibacterial activity and investigate the possible syner-
Introduction
gistic binding affinity that the coumarin and chalcone moieties pos-
sess when incorporated onto the same backbone using molecular
docking.
Coumarinyl chalcones are hybrid molecules combining the skeletal
frameworks of coumarins (aromatic lactones) and chalcones (aro-
matic α,β-unsaturated carbonyl compounds). Both these skeletal
frameworks are found abundantly in nature and may have a role
to play in the medicinal effects of the extracts of plants.[1–4] Because
both coumarins and chalcones each have a variety of medicinal
properties,[5–9] combining both these pharmacophores into one
molecule could result in either enhanced activity of each of their
properties individually or a broader spectrum of medicinal proper-
ties than either of them on their own.
These hybrid heterocyclic molecules were first synthesized from
3-acetyl coumarin and substituted benzaldehydes[10] by the
Claisen–Schimdt condensation carried out in an alcoholic solvent
and using either an ionic or organic base.[11,12] This has been a pop-
ular method for synthesizing coumarinyl chalcones;[13–15] however,
variations such as microwave solvent free synthesis have been car-
ried out with decreased reactions times and optimized yields.[16] Al-
though the preferred method has been to use bases such as
sodium hydroxide and piperidine, a greener approach involving
cellulose sulphonic acid as a catalyst was also employed.[17]
Hybrid coumarinyl chalcones have recently shown anticancer,[18]
antioxidant,[19] analgesic,[20] antiinflammatory[20] and antibacterial
activity[19–21] and when complexed with Ni and Cu together with
fluoroquinolones, played a part in developing highly active antican-
cer and antioxidant complexes.[22] This is not surprising, because
the two pharmacophores individually have shown activity in all
these assays.[23–28]
Experimental
General experimental procedures
1H and 13C NMR spectra were recorded at 298 K with 5–10 mg sam-
ples dissolved in 0.5 ml CDCl3 in 5 mm NMR tubes using a Bruker
AvanceIII 400 MHz NMR spectrometer (9.4T; Bruker, Germany)
(400.22MHz for 1H and 100.63 MHz for 13C. The digital digitizer res-
1
olution was set at 22 for both the H and 13C NMR experiments.
Chemical shifts (δ) are reported in parts per million and coupling
constants (J) in hertz. The 1H and 13C chemical shifts of the deuter-
ated solvent were 7.24 and 77.0 respectively, referenced to the in-
ternal standard, TMS. For the 1H NMR analyses, 16 transients were
acquired with a 1 s relaxation delay using 32 K data points. The
90° pulse duration was 10.0μs, and the spectral width was
8223.68 Hz. The 13C NMR spectra were obtained with a spectral
width of 24038.46 Hz using 64 K data points. The 90° pulse duration
was 8.40 μs. For the two-dimensional experiments including COSY,
NOESY, HSQC and HMBC, all data were acquired with 4 K × 128 data
points (t2 × t1). The mixing time for the NOESY experiment was 0.3 s,
and the long-range coupling time for the HMBC experiment was
65 ms. All data were analysed using Bruker Topspin 2.1 (2008)
software.
Reagent grade chemicals used in this study were purchased
from Sigma Aldrich, South Africa. IR spectra were recorded on
a Perkin Elmer Spectrum 100 FTIR spectrometer with universal
ATR sampling accessory. For GC-MS, the samples were analysed
on an Agilent GC-MSD apparatus equipped with DB-5SIL MS
(30 × 0.25 mm i.d., 0.25 μm film thickness) fused-silica capillary
To our knowledge, there have been no reports on docking stud-
ies with coumarinyl chalcones to any of the known protein targets
which we have used in this work; however, chalcones themselves
have been docked to the HIV-integrase enzyme[29] and the colchine
region of tubulin,[30] where the carbonyl group was found to be es-
sential for docking. Coumarins have been docked to the COX-1 and
COX-2 enzymes[31] and the metabolic enoyl-ACP-reductase enzyme
of bacteria.[32] There are no reports of coumarinyl chalcones being
docked to the penicillin binding protein 2X (PBP 2X) used in this
study.
*
Correspondence to: Neil A. Koorbanally, School of Chemistry and Physics,
University of KwaZulu-Natal, Private Bag X54001, Durban 4000, South Africa. E-
mail: Koorbanally@ukzn.ac.za
We herein report the synthesis and antibacterial activity of
coumarinyl chalcones and explore the effect that chloro, fluoro, hy-
droxy, methoxy and prenyl groups have on activity as well as deter-
mine which of the 2 or 4 positions were better for substitution with
a School of Chemistry and Physics, University of KwaZulu-Natal, Durban, South
Africa
b Discipline of Pharmaceutical Science, School of Health Science, University of
Kwazulu-Natal, Durban, South Africa
Magn. Reson. Chem. 2016, 54, 610–617
Copyright © 2016 John Wiley & Sons, Ltd.

The synthesis, structural elucidation and antimicrobial activity of 2- and 4-substitued- coumarinyl chalcones
column. Helium (at 2 ml minÀ1) was used as a carrier gas. The MS
was operated in the EI mode at 70 eV. Melting points were re-
corded on an Ernst Leitz Wetzlar micro-hot-stage melting point
apparatus. HRMS was carried out on a Bruker microTOF-Q II ESI
instrument operating at ambient temperatures, with a sample
concentration of 1 ppm. Column chromatography was carried
out on a 2 cm column with silica gel as the stationary phase
and varying solvent ratios of ethyl acetate and hexane as the
mobile phase. Samples of 60 ml were collected for each fraction
and masses typically between 0.5 and 1.0 g were loaded onto
the column.
(=CH), 129.7 (C-1), 131.9 (2C, C-2/6), 139.0 (=C), 163.85 (C-4), 190.8
(CHO).
Preparation of 3-acetyl-2H-chromen-2-ones (3a–l)
The synthesized 3-acetyl coumarin (5.31mmol, 1.00g) was added
to absolute ethanol (50 ml) with stirring, followed by the addition
of benzaldehydes 2a–l (10.62 mmol). A catalytic amount of piperi-
dine (10 drops) was then added and the reaction left to reflux for
5 h. On cooling, a yellow precipitate formed, which was then filtered
under vacuum and recrystallized in absolute ethanol to yield com-
pounds 3a–l with purities of between 78 and 99%.
(E)-3-(3-phenyl)-prop-2-enoyl)-2H-chromen-2-one (3a) light yellow
solid; 90% yield; mp 130–132 °C; UV λmax nm (log ε): 297 (2.86),
333 (2.69); IR υmax (cmÀ1): 1725 (O–C=O), 1656 (C=O), 1555 (C=C);
EIMS m/z (rel. int.) 276 (100) [M+], 248 (39), 231 (20), 131 (60), 103
(96), 77 (62).
(E)-3-(3-(2-methoxyphenyl)-prop-2-enoyl)-2H-chromen-2-one (3b)
bright yellow solid; 80% yield; mp 126–128 °C; UV λmax nm (log ε):
303 (3.30), 347 (3.32); IR υmax (cmÀ1): 1714 (O–C=O), 1654 (C=O),
1552 (C=C); EIMS m/z (rel. int.) 306 (12) [M+], 275 (100), 247 (4),
231 (11), 161 (11), 131 (3).
Synthesis
Chalcone (1,3-diphenylprop-2-enone) was also prepared according
to a published method in the literature because we wanted to com-
pare the activity results with this basic skeleton. The structure was
verified by NMR spectroscopy according to an authentic sample
available in the laboratory.[33]
Preparation of 3-acetyl-2H-chromen-2-one (1)
(E)-3-(3-(4-methoxyphenyl)-prop-2-enoyl)-2H-chromen-2-one (3c)
bright yellow solid; 85% yield; mp 130–132 °C; UV λmax nm (log ε):
280 (4.64), 372 (4.98); IR υmax (cmÀ1): 1717 (O–C=O), 1658 (C=O),
1555 (C=C); EIMS m/z (rel. int.) 306 (100) [M+], 278 (15), 263 (20),
161 (45), 133 (45), 108 (32).
(E)-3-(3-(2,4-dimethoxyphenyl)-prop-2-enoyl)-2H-chromen-2-one
(3d ) bright yellow solid; 93% yield; mp 150–153 °C; UV λmax nm (log
ε): 303 (3.06), 372 (3.21); IR υmax (cmÀ1): 1711 (O–C=O), 1654 (C=O),
1552 (C=C); EIMS m/z (rel. int.) 337 (11) [M+], 336 (56), 305 (100), 277
(2), 191 (23), 148 (27).
2-Hydroxybenzaldehyde (83.14 mmol, 10.07 g) and ethylaceto-
acetate (77.07 mmol, 10.03 g) was added to a solution of absolute
ethanol (50.0 ml). A catalytic amount of piperidine (10 drops) was
then added dropwise with cooling. The reaction was left to stir for
16 h at room temperature. The resulting precipitate was filtered
and recrystallized with glacial acetic acid to produce a light yellow
1
solid precipatate in 90% yield. H NMR (400 MHz, CDCl3): δ 2.71
(CH3), 7.33 (m, H-6′/8′), 7.62 (m, H-5′/7′), 8.47 (s, H-4′). 13C NMR
(100 MHz, CDCl3): δ 30.5 (CH3), 116.6 (C-8′), 118.3 (C-4a′), 124.5 (C-
3′), 125.0 (C-6′), 130.2 (C-5′), 134.4 (C-7′), 147.5 (C-4′), 155.3 (C-8a′),
159.3 (C-2′), 195.5 (C-9′).
(E)-3-(3-(2-chlorophenyl)-prop-2-enoyl)-2H-chromen-2-one
(3e)
light yellow solid; 78% yield; mp 140–142 °C; UV λmax nm (log ε):
305 (4.14), 332 (4.15); IR υmax (cmÀ1): 1718 (O–C=O), 1685 (C=O),
1555 (C=C); EIMS m/z (rel. int.) 275 [M+ À Cl] (100), 247 (8), 231
(12), 165 (7), 137 (19), 101 (32).
Preparation of (3-methylbut-2-enyloxy)benzaldehyde
intermediates (2k and 2l)
(E)-3-(3-(4-chlorophenyl)-prop-2-enoyl)-2H-chromen-2-one
(3f)
light yellow solid; 82% yield; mp 153–155 °C; UV λmax nm (log ε):
300 (4.34), 333 (4.39); IR υmax (cmÀ1): 1714 (O–C=O), 1666 (C=O),
1557 (C=C); EIMS m/z (rel. int.) 310 (100) [M+], 282 (76), 275 (23),
266 (14), 247 (38), 165 (57), 137 (76), 101 (92).
(E)-3-(3-(2-fluorophenyl)-prop-2-enoyl)-2H-chromen-2-one (3g) yel-
low solid; 80% yield; mp 153–155 °C; UV λmax nm (log ε): 328 (4.34),
370 (4.43); IR υmax (cmÀ1): 1722 (O–C=O), 1682 (C=O), 1557 (C=C);
EIMS m/z (rel. int.) 294 (80) [M+], 266 (73), 249 (8), 149 (69), 121
(76), 101 (100); HRMS: [M+ + Na] at m/z 317.0596 (Calculated for
C18H11O3FNa, 317.0590).
(E)-3-(3-(4-fluorophenyl)-prop-2-enoyl)-2H-chromen-2-one (3h) yel-
low solid; 80% yield; mp 142–145 °C; UV λmax nm (log ε): 300 (4.38),
350 (4.38); IR υmax (cmÀ1): 1715 (O–C=O), 1676 (C=O), 1556 (C=C);
EIMS m/z (rel. int.) 294 (80) [M+], 266 (73), 265 (40), 249 (19), 149
(69), 121 (76), 101 (100).
2-Hydroxybenzaldehyde or 4-hydroxybenzaldehyde (8.19 mmol,
1.00 g) was added to a solution of dry acetone (100 ml) followed
by the addition of anhydrous potassium carbonate (3.05 g).
1-Bromo-3-methyl-2-butene (prenyl bromide) (16.77 mmol,
2.5 g) was then added dropwise, and the reaction stirred at
room temperature for 3 h. On completion, the acetone was re-
moved under reduced pressure and the resulting crude solid
washed with water and extracted with 3 × 20 ml portions of diethyl
ether. The diethyl ether extract was concentrated and purified
using column chromatography with 8:2 hexane: ethyl acetate to
yield 0.75 g of 2k and 1.05 g of 2l respectively.
2-(3-methylbut-2-enyloxy)benzaldehyde (2k) brown oil (50%
1
yield); H NMR (400 MHz, CDCl3) δ 1.73 (CH3), 1.78 (CH3), 4.61 (d,
J =6.0 Hz, CH2), 5.47 (t, J = 6.0Hz, =CH), 6.97 (2H, m, H-3/5), 7.50 (t,
7.0 Hz, H-4), 7.80 (d, J= 7.0 Hz, H-6), 10.4 (s, CHO); 13C NMR
(100 MHz, CDCl3): δ 18.4 (CH3), 26.0 (CH3), 65.5 (CH2), 113.1 (C-3),
119.1 (=CH), 120.5 (C-5), 125.1 (C-1), 128.3 (C-4), 135.9 (C-6), 138.6
(=C), 161.5 (C-2), 190.0 (CHO).
4-(3-methylbut-2-enyloxy)benzaldehyde (2l) brown oil (60% yield);
1H NMR (400 MHz, CDCl3): δ 1.73 (CH3), 1.78 (CH3), 4.57 (d, J = 6.0Hz,
CH2), 5.47 (t, J = 6.0Hz, =CH), 6.98 (d, J = 8.0 Hz, 2H, H-3/5), 7.79 ( d,
J =8.0 Hz, 2H, H-2/6), 9.85 (s, CHO); 13C NMR (100 MHz, CDCl3): δ
18.05 (CH3), 25.64 (CH3), 65.1 (CH2), 115.0 (2C, C-3/5), 118.79
(E)-3-(3-(2-hydroxyphenyl)-prop-2-enoyl)-2H-chromen-2-one (3i)
yellow solid; 75% yield; mp 158–161 °C; UV λmax nm (log ε): 247
(4.01), 342 (4.39); IR υmax (cmÀ1): 3341 (OH), 1700 (O–C=O), 1651
(C=O), 1571 (C=C); EIMS m/z (rel. int.) 292 (100) [M+], 264 (30), 247
(20), 147 (61), 119 (46), 91 (68).
(E)-3-(3-(4-hydroxyphenyl)-prop-2-enoyl)-2H-chromen-2-one (3j)
yellow solid; 88% yield; mp 188–190 °C; UV λmax nm (log ε): 310
(4.28), 360 (4.45); IR υmax (cmÀ1): 3294 (OH), 1698 (O–C=O), 1650
Magn. Reson. Chem. 2016, 54, 610–617
Copyright © 2016 John Wiley & Sons, Ltd.
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T. Moodley et al.
Scheme 1. Synthesis of 2- and 4- substituted coumarinyl chalcones.
(C=O), 1550 (C=C); EIMS m/z (rel. int.) 292 (100) [M+], 264 (25), 247
(18), 147 (46), 119 (43), 91 (54).
(E)-3-(3-(2-(3-methylbut-2-enyloxy)phenyl)-prop-2-enoyl)-2H-chro-
men-2-one (3k) yellow solid; 80% yield; mp 92–95 °C; UV λmax nm
(log ε): 312 (4.35), 363 (4.46); IR υmax (cmÀ1): 1727 (O–C=O), 1606
(C=O), 1559 (C=C); EIMS m/z (rel. int.) 360 (19) [M+], 327 (15), 173
(100), 146 (19); HRMS: [M+ + Na] at m/z 383.1268 (Calculated for
were standardized using a 0.5 McFarland standard turbidity and
then swabbed onto agar plates. Paper discs were dissolved in sam-
ple at concentrations of between 99 and 101μg for the coumarinyl
chalcones and chalcone itself and at 99μg for 3-acetylcoumarin
and placed onto the prepared agar plates which were inverted
and incubated at 35–37 °C for 24 h. The diameter of the zone of in-
hibition was measured in millimetres. Compounds 3c–e, 3g and 3i
as well as chalcone showed inhibition zones of >3 mm and were
selected to determine their minimum bactericidal concentration
(MBC) values using the broth microdilution assay with ampicillin
as the control and following the method in Andrews.[34]
C23 H20O4Na, 383.1259).
(E)-3-(3-(4-(3-methylbut-2-enyloxy)phenyl)-prop-2-enoyl)-2H-chro-
men-2-one (3l) yellow solid; 89% yield; mp 119–121 °C; UV λmax nm
(log ε): 295 (4.01), 376 (4.21); IR υmax (cmÀ1): 1719 (O–C=O), 1660
(C=O), 1567 (C=C); EIMS m/z (rel. int.) 360 (10) [M+], 292 (100), 173
(34), 147 (40), 67 (50); HRMS: [M+ + Na] at m/z 383.1269 (Calculated
for C23H20O4Na, 383.1259).
Broth dilution method
Compounds 3c–3e, 3g and 3i and chalcone was dissolved in DMSO
(10 mg mlÀ1) and serially diluted 5 times with Mueller–Hinton
broth, inoculated with the respective bacterial cultures and incu-
bated at 37 °C for 18 h. This was performed in duplicate. Thereafter,
10μl from each concentration was placed on Mueller–Hinton plates
and incubated at 37 °C for 18 h to determine the MBC. The MBC was
the lowest concentration, which showed no bacterial growth in the
area in which the sample was placed.
Antibacterial assay
Microbial strains
The antimicrobial activity of the synthesized compounds 3a–3l, its
precursor, 3-acetylcoumarin and chalcone (1,3-diphenylprop-
2-enone) were tested against two Gram-positive bacteria, Staphylo-
coccus aureus 29263 and S. aureus Rosenbach ATCC BAA-1683
(methicillin resistant S. aureus) and two Gram-negative bacteria,
Klebsiella pneumonia ATCC 31488 and Escherichia coli ATCC 25922
as well as one fungal species, Candida albicans ATCC 10231, accord-
ing to the disc diffusion method.
Molecular docking studies
The crystal structure of Cefditoren (CDS) bound to the active site
of PBP 2X was obtained from the Protein Data Bank (http://www.
rscb.org/pdb) with the PDB ID: 2Z2M. Co-crystallized ligands
were identified and removed from the target proteins, and crys-
tallographic water molecules were eliminated from the 3D coor-
dinate file. A geometry optimization of all the compounds was
performed by Gaussian 2009 software[35] by the semiempirical
method PM6 for flexible conformation of the compounds during
docking. A grid box (including residues within a 10.0 Å radius)
large enough to accommodate the active site was constructed
Disc diffusion method
The standard antibiotics ciprofloxacin and ampicillin were used as
controls for comparison. Mueller Hilton agar was prepared (38 g in
1 l of water), poured into sterile prelabeled petri dishes and allowed
to set and dry at room temperature. Bacterial or fungal organisms
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Copyright © 2016 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2016, 54, 610–617

The synthesis, structural elucidation and antimicrobial activity of 2- and 4-substitued- coumarinyl chalcones
Magn. Reson. Chem. 2016, 54, 610–617
Copyright © 2016 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/mrc

T. Moodley et al.
Table 2. 13C NMR data (δ in ppm) of coumarinyl chalcones 3a–l (140 MHz, CDCl3, J is given in Hz)
No.
3a
3b
3c
3d
3e
3f
3g
3h
3ia
3ja
3k
3l
OCH3
2′
—
55.9
159.2
125.7
147.6
118.6
129.4
124.8
134.0
116.7
155.2
186.9
124.3
140.4
123.7
159.0
111.1
132.2
120.7
129.9
—
55.4
159.3
127.6
147.8
118.6
129.9
124.9
134.0
116.7
155.1
186.3
121.6
145.0
125.5
130.8
114.4
161.9
114.4
130.8
—
55.5 (4) 55.7 (2)
159.4
126.2
147.4
118.7
129.9
124.8
133.8
116.6
155.1
186.8
121.9
140.7
117.2
160.6
98.4
—
—
—
—
—
—
—
—
159.3
125.3
148.1
118.5
130.0
125.0
134.2
116.7
155.2
186.5
123.9
145.1
134.8
128.9
128.9
130.8
128.9
128.9
—
159.4
125.0
148.4
118.5
130.2
125.0
134.4
116.8
155.3
186.2
126.2
140.5
133.0
135.8
130.1
131.4
127.1
128.1
—
159.4
125.2
148.4
118.6
130.1
125.2
134.3
116.7
155.3
186.3
124.4
143.5
133.2
129.9
129.2
136.7
129.2
129.9
—
159.2
159.3
158.4
125.6
146.8
118.3
130.3
124.9
134.1
116.2
154.4
187.3
123.8
139.8
121.1
157.4
116.1
132.3
119.4
128.9
—
158.3
125.8
146.3
118.3
130.2
125.4
133.9
116.1
154.3
187.0
121.2
144.9
124.9
130.9
115.9
160.4
115.9
130.9
—
159.1
125.8
147.5
118.5
129.8
124.8
133.9
116.6
155.1
187.1
124.2
140.8
124.1
158.4
112.5
132.0
120.6
129.4
65.6
159.4
125.5
147.8
118.6
130.0
124.9
134.1
116.6
155.2
186.3
121.5
145.3
127.5
130.9
115.1
161.4
115.1
130.9
65.0
3′
125.2
125.1
4′
148.3
148.2
4a′
5′
118.5
118.5
130.1
130.0
6′
125.0
125.0
7′
134.3
134.4
8′
116.7
116.7
8a′
9′
155.2
155.2
186.5
186.3
10′
11′
1
125.9 (d, 6.7)
137.1
123.8 (d, 2.9)
143.7
122.9 (d, 12.4)
161.8 (d, 252.5)
116.1 (d, 19.4)
132.2 (d, 8.8)
124.4 (d, 4.5)
129.2 (d, 3.0)
—
131.0 (d, 3.2)
130.8 (d, 10.4)
116.1 (d, 19.5)
164.3 (d, 257.3)
116.1 (d, 19.5)
130.8 (d, 10.4)
—
2
3
4
163.5
105.5
131.2
—
5
6
1″
2″
3″
4″
5″
—
—
—
—
—
—
—
—
—
—
119.6
137.9
18.4
119.1
138.8
18.2
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
25.9
25.8
aData acquired in DMSO-d6.
to identify other residual interactions of the test compounds. An
essential feature of the binding site is hydrogen bonding and ar-
omatic π–π stacking interactions. The 2D structures of the mole-
cules were converted to energy minimized 3D structures for in
silico protein–ligand docking calculations.
such as piperidine removes the alpha proton of the methy-
lene group between the two carbonyl groups, which make
it very acidic. The carbonyl group of the ester then un-
dergoes enolisation, which results in the nucleophilic attack
of the carbonyl group of benzaldehyde leading to lactone
and coumarin formation. The coumarin intermediate was pro-
duced in a yield of 90%. In the second step, 3-acetylcoumarin
underwent
a Claisen–Schmidt condensation with chloro,
Results and Discussion
fluoro, hydroxy and methoxy benzaldehydes 2a–j to produce
the coumarinyl chalcones 3a–j. All compounds were yellow
precipitates with yields in excess of 80%. The 2-fluoro
coumarinyl chalcone 3g and the 2-O-prenylated and 4-O-
prenylated coumarinyl chalcones, 3k and 3l were synthesized for
the first time in this work. For the novel prenylated coumarinyl
chalcones 3k and 3l, 2- and 4-hydroxybenzaldehdyes were each
Synthesis
Coumarinyl chalcones 3a–l were synthesized in a two-step reaction,
which involved the synthesis of 3-acetylcoumarin, which was
synthesized by the Knoevenagel condensation of 2-hydroxy-
benzaldehyde and ethyl acetoacetate (Scheme 1). A mild base
Table 3. Minimum bactericidal concentrations (MBC) of the test compounds (mM) against two gram +ve and two gram Àve bacterial species and
a Candida species
Compound
Substitution
S. aureus
MRSA
0.0018
K. pneumonia
E. coli
0.0018
C. albicans
Ciprofloxacin
-
-
0.0018
0.037
0.0074
Ampicillin
0.056
0.89
0.89
0.89
1.8
9
-
Chalcone
-
9
33
-
18
-
-
-
-
-
-
-
-
-
-
-
-
-
3c
3d
3e
3g
3i
4-OCH3
4-Cl
2-F
-
32
-
34
32
-
-
2-OH
2-OPr
-
-
27
-
Hyphen denotes no activity at the highest concentration tested.
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Magn. Reson. Chem. 2016, 54, 610–617

The synthesis, structural elucidation and antimicrobial activity of 2- and 4-substitued- coumarinyl chalcones
Table 4. Docking scores of 3a–l against trypsin-digested PBP 2X. Binding energies are calculated using Autodock
Compound (Autodock) Binding
Intermolecular
Number of
Residues involved in
Active residues
near the gate of
active site of PBP2X
energy Kcal molÀ1 energy Kcal molÀ1 hydrogen bonds hydrogen bonding
3a
3b
3c
3d
3e
3f
À5.75
À6.32
À6.20
À6.65
À5.75
À5.60
À5.99
À5.80
À6.90
À6.44
À7.74
À7.73
À8.04
À8.73
À9.22
À9.18
À9.26
À8.73
À8.58
À8.97
À8.78
À9.88
À9.43
À10.56
À10.52
À10.13
2
2
2
3
2
2
2
2
3
3
2
2
6
ASN397 GLN552
ASN397 THR550
ASN397 GLN552
Gln552 Arg372 ASN397
ASN397 THR550
ASN397 GLN552
ASN397 THR550
ASN397 GLN552
Ala551, Ser337, Ser558, Thr550, Gly336, Phe550
Asp373, Ser395, Asn397, Gly336, His394, Trp374
Gln452, Thr550, Glu378, Trp374, Tyr561
Gly336, Gly451 Thr550
Ser395, Gly549, Tyr561, Gly451, Ser548,
Ser337, Thr550, Ala551, Phe450, Gln452, Ser395,
Ser548,Ser395, Trr561,Phe450, Glu452, Gly549.
Ser337, Ser548, Phe450, Ala551, Thr550, Gly549
3g
3h
3i
ASN397 THR550 GLN552 Ser395, Ser 548, Ser337, Gly451
3j
SER337 TRP374 GLN552
ASN397 GLN552
Lys340, Asp375, Asp373
3k
3l
AMa
Gln452, Thr370, Arg372, Glu334, Thr550, Asp373
His394, Ser337, Glu334, Gln552, Thr550, Trp374
Thr526, Asn397, Trp374, Asp373,
SER337 SER395
Glu378, Asn377, Thr550,
Ser337, Ser395, Ser548
Thr550, Ser395, ser337,
Gln452, Glu334, Gln552.
CFDb
À8.75
À11.44
6
Ser548, Gly549, Tyr561, Ala551, Thr526.
aAmoxicillin.
bCefditoren.
treated with prenyl bromide to form the O-prenylated inter-
mediates, 2k and 2l before being condensed with 3-acetyl
coumarin. Potassium carbonate was used as a base to abstract
the hydroxyl proton and to neutralize the HBr formed during
the reaction.
BAA-1683), the latter of which was methicillin resistant S. aureus,
and two gram negative bacteria, K. pneumonia ATCC 31488 and
E. coli ATCC 25922 as well as one fungal species, C. albicans ATCC
10231. Along with these compounds, chalcone (1,3-diphenylprop-
2-enone) and the precursor, acetylcoumarin were also tested in
the same assays for comparison in order to see whether adding a
coumarin moiety to the chalcone increased activity or whether
the chalcone moiety had a positive influence on the antibacterial
action of the compounds. The results are contained in Table 3.
In general, the compounds were not as active as the ampicillin
and ciprofloxacin standards; however, chalcone showed bacteri-
cidal activity of between 9 and 18 mM and five of the coumarinyl
chalcones showed bactericidal activity of between 27 and 34 mM
in at least one of the Staphyloccus species or C. albicans. The com-
pounds were totally inactive to the Gram-negative species.
Chalcone itself was the most active and showed the broadest spec-
trum of activity, being active against both Staphyloccus species and
C. albicans. The activity of chalcone against MRSA was 20-fold lower
than that of ampicillin. In the fungal species, chalcone showed a
fivefold lower activity than ampicillin.
The coumarin moiety did not enhance the activity of chalcone. In
fact, acetylcoumarin itself was totally inactive to all the microbial
species tested against. Adding particular substituted chalcone
moieties to this increased the activity somewhat. In particular, the
4-OCH3, 2-F and 2-OH (3c, 3e and 3g) derivatives were now active
against S. aureus ATCC 29263 between 32 and 34 mM and 3i (the
O-prenylated derivative) was active against MRSA at 27 mM. The 4-
Cl derivative (3d) was the only coumarinyl chalcone to show any ac-
tivity against Candida. These results also indicated that the type and
position of the substituent were important for antibacterial activity.
Structural elucidation
The acetylcoumarin intermediate was identified by its character-
istic H-4′ singlet resonance at δ 8.47, a lactone carbonyl reso-
nance at δ 159.3 (C-2′) and aromatic proton resonances at δ
7.29 to 7.64 (H-5′–H-8′). The carbon resonances for the acyl
group present occurred at δ 195.5 (C-9′) and δ 30.5 (CH3). Forma-
tion of the chalcone was indicated by a pair of doublets in the re-
gion between δ 7.81 and δ 8.29. In the case of compounds 3c, 3g
and 3l, the H-10′ and H-11′ resonances had almost similar chem-
ical shifts in CDCl3 because of the electronic effects of the differ-
ent substituents. To explore this further, we acquired a NMR
sample of 3c in DMSO-d6 to see if these proton resonances
would separate. Interestingly, they did. These resonances now
separated at δ 7.51 (H-10′) and δ 7.73 (H-11′), indicating that
these protons were able to interact with the solvent. Resonance
effects of the α,β-unsaturated carbonyl group indicated that H-
10′ is more shielded than H-11′; however, we have observed that
in three of the compounds, 3a (unsubstituted), and the two para
substituted halogenated compounds, 3f (4-Cl) and 3h (4-F), the
H-10′ resonance appears downfield to H-11′. In all other com-
pounds, H-10′ is more shielded which are consistent with reso-
nance effects.
The full structural elucidation of all the synthesized molecules
3a–3l is given in Tables 1 and 2.
Molecular docking studies
Molecular docking analysis for the synthesized coumarinyl
chalcones 3a–3l was carried out with the crystal structure of
cefditoren (CDS) bound to the active site of PBP 2X to identify the
variations in binding affinity using Autodock4 software. The crystal
Anti-bacterial activity
Compounds 3a–3l were tested for their antibacterial activity
against two gram positive, S. aureus species (ATCC 29263 and ATCC
Magn. Reson. Chem. 2016, 54, 610–617
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T. Moodley et al.
structure of this complex was identified in the Protein data bank as
PDB ID: 2Z2M. The PDB structure 2Z2M bound to the inhibitor CDS
shows a true binding site for each of the subunits and was con-
sidered as the centre of search space for docking. The molecular
docking results are summarized in Table 4. For the test compounds
unsaturated carbonyl hydrogen bonding to Asn 397, the oxygen
of the coumarin hydrogen bonding to Thr 550 and the 2-OH group
to Gln 552. Compound 3k fits nicely into the pocket in the enzyme
with hydrogen bonding from the α,β-unsaturated carbonyl to Asn
397 and from the heteratomic oxygen of the coumarin to Gln
552. Hydrogen bonding between the α,β-unsaturated carbonyl
group with the Asn 397 residue could be a reason why the chalcone
scaffolds are more active than the just the coumarin alone.
3a to 3l, the Autodock score ranged from À5.60 to À7.74 Kcal molÀ1
,
while the Autodock score of the standard drugs Amoxicillin and CDS
was À8.04 and À8.75 Kcal molÀ1 respectively. Based on the
Autodock score, compounds 3i (2-hydroxycoumarinyl chalcone), 3k
(2-oxyprenylcoumarinylchalcone) and 3l (4-oxyprenylcouma-
rinylchalcone) with Autodock scores of À6.90, À7.74 and
À7.73 Kcal molÀ1 respectively showed the best binding affinity using
the criteria that the lower the binding energy score the higher the
binding affinity.
Conclusion
Claisen–Schmidt condensation was used to synthesize 12
coumarinyl chalcones from coumarinyl acetophenone and benzal-
dehyde precursors. The substituents on the benzaldehdye derived
aromatic ring were hydroxy, methoxy, chloro, fluoro and prenyloxy
groups, which were substituted at the 2 and 4 positions. The 1H and
13C NMR resonances of all synthesized compounds were unambig-
uously assigned and will provide a basis for the structural determi-
nation of similar compounds. Two of the compounds in the series,
3k (2-oxyprenylcoumarinyl chalcone) and 3l (4-oxyprenylcouma
rinyl chalcone) were novel derivatives that showed a binding affin-
ity close to the standard amoxicillin and CDS. The coumarinyl moi-
ety of the chalcone did not enhance the antimicrobial activity of
chalcone, but instead led to worse activity.
The mode of interaction between all docked synthesized com-
pounds and the trypsin-digested PBP 2X receptor were created
with Ligplot software. Compound 3k with the best binding affinity
docked in its best conformation into the binding site of trypsin-
digested PBP 2X is shown in Fig. 1. This model was considered as
a clue to obtaining a clear picture of the relative arrangement of
binding interaction sites. The hydrogen bond interactions with
the residues (in green) are shown by dashed lines, and hydrophobic
contacts are represented by an arc with spokes radiating toward
the ligand atoms they come into contact with. As can be seen from
Fig. 2 compound 3i fits nicely into the enzyme, with the α,β-
Figure 1. Compound 3k docked in its best conformation into the binding site of trypsin-digested PBP 2X; Hydrogen bonds are represented by broken lines
(blue). (A) Binding mode of compound 3k showing interactions with the protein, (B) 3k fitting into the binding pocket of the protein.
Figure 2. Schematic diagrams of protein-ligand interactions. (A) Structure of docked ligand 3i-2Z2M complex (B) Structure of docked ligand 3k-2Z2M
complex.
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Magn. Reson. Chem. 2016, 54, 610–617

The synthesis, structural elucidation and antimicrobial activity of 2- and 4-substitued- coumarinyl chalcones
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