Benzofuranyl esters: Synthesis, crystal structure determination, antimicrobial and antioxidant activities

Article abstract of DOI:10.3390/molecules200916566

A series of five new 2?(1?benzofuran?2?yl)?2?oxoethyl 4-(un/substituted)benzoates 4(a-e), with the general formula of C₈H₅O(C=O)CH₂O(C=O)C₆H₄X, X = H, Cl, CH₃, OCH₃ or NO₂, was synthesized in high purity and good yield under mild conditions. The synthesized products 4(a-e) were characterized by FTIR, ¹H-, ¹³C- and ¹H-¹³C HMQC NMR spectroscopic analysis and their 3D structures were confirmed by single-crystal X-ray diffraction studies. These compounds were screened for their antimicrobial and antioxidant activities. The tested compounds showed antimicrobial ability in the order of 4b < 4a < 4c < 4d < 4e and the highest potency with minimum inhibition concentration (MIC) value of 125 μg/mL was observed for 4e. The results of antioxidant activities revealed the highest activity for compound 4e (32.62% ± 1.34%) in diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, 4d (31.01% ± 4.35%) in ferric reducing antioxidant power (FRAP) assay and 4a (27.11% ± 1.06%) in metal chelating (MC) activity.

Full text of DOI:10.3390/molecules200916566

Molecules 2015, 20, 16566-16581; doi:10.3390/molecules200916566
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Benzofuranyl Esters: Synthesis, Crystal Structure
Determination, Antimicrobial and Antioxidant Activities
C. S. Chidan Kumar 1,2,*, Li Yee Then 1, Tze Shyang Chia 1, Siddegowda Chandraju 3,
Yip-Foo Win 4, Shaida Fariza Sulaiman 5, Nurul Shafiqah Hashim 5, Kheng Leong Ooi 5,
Ching Kheng Quah 1,* and Hoong-Kun Fun 1,6
1
X-ray Crystallography Unit, School of Physics, Universiti Sains Malaysia, Penang 11800,
Malaysia; E-Mails: lyeeth0923@hotmail.com (L.Y.T.); chiatzeshyang@hotmail.com (T.S.C.);
hkfun@usm.my or hfun.c@ksu.edu.sa (H.-K.F.)
2
Department of Engineering Chemistry, Alva’s Institute of Engineering & Technology,
Visvesvaraya Technological University, Mijar, Moodbidri 574225, Karnataka, India
3
Department of Sugar Technology & Chemistry, University of Mysore, Sir M. V. PG Center,
Tubinakere, Mandya 571402, Karnataka, India; E-Mail: chandraju1@yahoo.com
4
Department of Chemical Science, Faculty of Science, Universiti Tunku Abdul Rahman,
Perak Campus, Jalan Universiti, Bandar Barat, Kampar 31900, Malaysia;
E-Mail: yipfw@utar.edu.my
5
School of Biological Sciences, Universiti Sains Malaysia, Penang 11800, Malaysia;
E-Mails: shaida@usm.my (S.F.S.); nshafiqah25@gmail.com (N.S.H.); ooilng@yahoo.com (K.L.O.)
6
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia
* Authors to whom correspondence should be addressed;
E-Mails: chidankumar@aiet.org.in (C.S.C.K.); ckquah@usm.my (C.K.Q.);
Tel.: +60-46-533-888 (ext. 3690) (C.S.C.K. & C.K.Q.);
Fax: +60-46-579-150 (C.S.C.K. & C.K.Q.).
Academic Editor: Derek J. McPhee
Received: 21 July 2015 / Accepted: 17 August 2015 / Published: 11 September 2015
Abstract: A series of five new 2‐(1‐benzofuran‐2‐yl)‐2‐oxoethyl 4-(un/substituted)benzoates
4(a–e), with the general formula of C8H5O(C=O)CH2O(C=O)C6H4X, X = H, Cl, CH3, OCH3
or NO2, was synthesized in high purity and good yield under mild conditions. The synthesized
products 4(a–e) were characterized by FTIR, 1H-, 13C- and 1H-13C HMQC NMR spectroscopic
analysis and their 3D structures were confirmed by single-crystal X-ray diffraction studies.

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These compounds were screened for their antimicrobial and antioxidant activities. The
tested compounds showed antimicrobial ability in the order of 4b < 4a < 4c < 4d < 4e and
the highest potency with minimum inhibition concentration (MIC) value of 125 µg/mL was
observed for 4e. The results of antioxidant activities revealed the highest activity for
compound 4e (32.62% ± 1.34%) in diphenyl-2-picrylhydrazyl (DPPH) radical scavenging,
4d (31.01% ± 4.35%) in ferric reducing antioxidant power (FRAP) assay and 4a (27.11% ±
1.06%) in metal chelating (MC) activity.
Keywords: benzofuran; spectroscopic analysis; antimicrobial; antioxidant; XRD; substituent
1. Introduction
Benzo[b]furan nucleus is widespread in plants and often the natural products possessing benzofuran
are useful for their immense pharmacological properties. Systematic investigation of this heterocyclic
compound plays important roles in the development of medicinal chemistry and synthetic products [1,2].
Pyromucic acid (2-furoic acid) is the first prepared furan compound discovered in 1780 [3]. Basically,
benzo[b]furan ring systems bearing various substituents at the C-2 position are broadly distributed and
had been reported to possess antifungal, antiviral and antioxidant activities [4,5]. Some 2-arylbenzofuran
derivatives are well-known biodynamic agents possessing a wide range of biological activities, including
calcium blockers, phytoestrogens, antioxidative, anticancer, insecticidal, antiproliferative, antiviral,
antifungal, antiplatelet, anti-inflammatory, immunosuppressive, antifeedant and cancer preventative
activity [6–18]. Besides the bioactivities, benzo[b]furan derivatives can act as building blocks for
fluorescent sensors [19] and optical brighteners.
Encouraged by the biological activities associated with the benzo[b]furan ring system, we herein
report the efficient synthesis, spectra, X-ray crystal structure analysis and biological activities of five
new benzo[b]furan esters 4(a–e).
2. Results and Discussion
2.1. Chemistry
Generally, the compounds 4(a–e) revealed absorption bands above 3000 cm−1 which indicate the
presence of unsaturated C-H (benzene and benzofuran) groups whereas the methyl group, -CH3, as well
as methylene group, -CH2- revealed asymmetric and symmetric C-H stretching frequencies around
2966–2858 cm−1 [20]. The infrared spectra studies of 4(a–e) also revealed the presence of ν(C=C)
bands which are usually found for benzene and benzofuran groups. In addition, the compounds 4(a–e)
revealed two distinct ν(C=O) bands in the range of 1725–1682 cm−1 in which the ν(C=O) band with
the lower wave number is attributed to the C=O of carboxylate anion [21–24]. Two exceptional cases
which are the presence of ν(C-Cl) at 1086 cm-1 in 4b and the presence of ν(NO2) at 1524 cm−1 in 4e
were observed.
The 1H-NMR spectra of compounds 4(a–e) exhibited similarities among each other with the presence
of -CH2- protons centering around δ ≈ 5.55 ppm and two sets of well-resolved doublet centering around

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δ ≈ 7.31 and 8.09 ppm with the integration values of 2:2, ascribed to the -CH- protons of benzene
group [20]. Compound 4e revealed one exceptional sharp singlet peak at 8.32 ppm with the integration
value of 4 indicating the total number of benzene protons. This peak is originated from the attached
NO2 group at the para position of benzene ring. In addition, all the five protons of benzofuran are
located in the downfield region in the 1H-NMR spectra centering around δ ≈ 7.33, 7.51, 7.57, 7.63 and
7.73 ppm with the integration values of 1:1:1:1:1. The exceptional and predictable observations are
the occurrence of -CH3 and -OCH3 proton signals of compounds 4c and 4d in the upfield region at
2.42 and 3.87 ppm, respectively. Based on the integration values, the number of protons in compounds
4(a–e) are in accordance with the number of protons proposed.
All compounds exhibited three distinct sets of carbon signals in the 13C-NMR spectra. In the downfield
13
region of C NMR spectra, both δ(C=O) and δ(COO) signals are located at δ ≈ 183.71 ppm and
δ ≈ 165.47 ppm, respectively, whereas the -CH2- carbon signals are located in the upfield region
centering around δ ≈ 66.42 ppm [22–24]. The exceptional and predictable observations are the occurrence
of -CH3 and -OCH3 carbon signals of compounds 4c and 4d, respectively, in the upfield region of
13C-NMR spectra. In the 13C-NMR spectra study, the carbon signals of benzene and benzofuran groups
were found in the range of 112.59–155.79 ppm [21–24]. The carbon signals centering at δ ≈ 112.60,
113.60, 123.62, 124.33, 126.98, 128.87, 150.63 and 155.79 ppm are attributed to benzofuran groups
and the remaining signals are attributed to benzene carbons.
2.2. X-ray Crystal Structure Description
2-(Benzofuran-2-yl)-2-oxoethyl 4-(un/substituted)benzoates with different functional groups viz., no
substitution (4a), -chloro (4b), -methyl (4c), -methoxy (4d) and -nitro (4e) substituted at the -para position
of phenyl ring were subjected to X-ray diffraction analysis. Crystal data and refinement parameters of
the analyzed compounds are listed in Table 1. The hydrogen bonds geometry and π···π interactions are
presented in Tables S1 and S2 (Supplementary Materials).
Table 1. Crystal data and refinement parameters for 4(a–e).
Compound
4a
1,037,756
C17H12O4
280.27
4b
1,037,759
C17H11ClO4
314.71
4c
1,037,762
C18H14O4
294.29
4d
4e
CCDC Deposition Number
1,037,763
C18H14O5
310.29
1,037,764
C17 H11NO6
325.27
Molecular Formula
Molecular Weight
Crystal System
Space Group
a (Å)
Monoclinic
P21/n
Monoclinic
C2/c
Monoclinic
P21/n
Triclinic
Triclinic
ꢁ
ꢀ1
ꢁ
ꢀ1
10.2230(9)
8.4353(7)
16.1205(15)
90
33.483(5)
5.3687(8)
26.404(4)
90
12.5266(13)
6.7689(7)
18.0335(19)
90
6.9335(6)
8.4724(7)
12.8702(11)
103.539(2)
95.789(2)
92.664(2)
729.36(11)
2
6.5424(10)
13.197(2)
16.973(3)
79.694(3)
87.211(3)
85.715(3)
1436.9(4)
4
b (Å)
c (Å)
α (°)
β (°)
98.300(2)
90
114.833(3)
90
108.165(2)
90
γ (°)
V (Å3)
1375.6(2)
4
4307.6(10)
12
1452.9(3)
4
Z
D
calc (g·cm−3)
1.353
1.456
1.345
1.413
1.504
Crystal Dimension (mm)
µ (mm−1)
0.25 × 0.36 × 0.48 0.08 × 0.17 × 0.46 0.14 × 0.24 × 0.46 0.20 × 0.27 × 0.41 0.12 × 0.20 × 0.41
0.10 0.28 0.10 0.10 0.12

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Table 1. Cont.
Compound
Tmin/Tmax
4a
0.901/0.976
15,170
−14, 9;
−9, 11;
−22, 22
2.2–30.2
4037
4b
0.857/0.977
17,994
−40, 40;
−6, 6;
4c
0.903/0.987
16,236
−17, 16;
−9, 9;
4d
0.899/0.980
14,866
−9, 9;
4e
0.903/0.986
37,178
−7, 7;
Reflections Measured
Indices Range (h, k, l)
−11, 11;
−18, 18
1.6–30.2
4283
−15, 15;
−20, 20
1.6–25.0
4946
−32, 32
1.7–26.0
4235
−25, 24
1.8–30.0
4234
θ Limit (°)
Unique Reflections
Observed Reflections (I > 2σ(I))
Parameters
2630
2044
2835
3078
3889
190
397
200
209
433
Goodness of Fit on F2
1.04
0.98
1.03
1.07
1.11
R1,wR2 [I > 2σ(I)]
0.047, 0.162
0.044, 0.143
0.045, 0.148
0.045, 0.149
0.065, 0.216
Molecular conformations of these compounds are characterized by three degree-of-freedom, which
are the O1–C8–C9–O3, C9–C10–O2–C11 and O2–C11–C12–C13 torsion angles or denoted hereafter as
τ1, τ2 and τ3, respectively (Figure 1). Basically, torsion angles τ1 and τ3 are close to 0° or ±180°,
indicating small deviations of the ethanone and carboxylate groups from attached benzofuran and phenyl
rings, respectively. Since these deviations are very small, the 1-(benzofuran-2-yl)ethanone group and
benzoate group are taken as two rigid bodies.
Figure 1. General chemical diagram of 4(a–e) shows τ1, τ2 and τ3 torsion angles.
The asymmetric units of compounds 4a, 4c and 4d consist of one unique molecule, while two
crystallographically independent molecules (molecules A and B) are observed in compounds 4b and 4e.
Basically, the compounds under study tend to adopt two types of conformations, in which the benzofuran
group is either nearly-planar or nearly-perpendicular with respect to the phenyl ring, as indicated by
the torsion angle τ2. Compounds 4a, 4c and 4d adopt nearly-perpendicular conformation with τ2 close
to 90°, 4e is a nearly-planar conformer with τ2 approaches 180° and interestingly, 4b exhibits both in
its crystal structure as summarized in Table 2. The overlay diagrams of both conformers are depicted in
Figures 2 and 3. Overall, the values of τ2 are still in good agreement with the conformation distribution
of phenacyl benzoate derivatives reported earlier [25] after the replacement of phenyl ring with
benzofuran ring.

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Table 2. Torsion angles τ1, τ2 and τ3 for 4(a–e).
Compound O1–C8–C9–O3 (τ1,°) C9–C10–O2–C11 (τ2,°) O2–C11–C12–C13 (τ3,°)
4a
4b
4c
4d
4e
−176.92(14)
5.3(4), −175(2)
−3.5(2)
75.19(17)
162.5(2), −70.2(13)
78.36(16)
171.53(13)
−176.7(2), −172(2)
−171.25(12)
−2.62(19)
79.49(15)
−169.34(11)
7.2(4), 2.5(4)
177.4(2), 179.6(3)
−175.4(3), −175.6(3)
Figure 2. Overlay diagram of 4a, 4b (inverted molecule B), 4c and 4d shows similar,
nearly-perpendicular conformation.
Figure 3. Overlay diagram of nearly flat conformer, 4b (molecule A), 4e (molecules A and
B), viewed (up) perpendicular to or (down) along the mean plane.
In compound 4a, molecules are linked by weak intermolecular C10–H10A···O4 hydrogen bonds,
involving the methylene and carbonyl groups, into zero-dimensional dimeric structures (Figure 4) with
R22 (10) graph-set motif. This hydrogen-bonded structure is also observed in other related structures
such as 2-oxo-2-phenylethyl benzoate [26], 2-(4-bromophenyl)-2-oxoethyl 2-aminobenzoate [25] and
2-(4-bromophenyl)-2-oxoethyl 4-methoxybenzoate [27], suggesting this dimeric form is a favoured
interaction pattern in compound with nearly-perpendicular conformation. Weak intermolecular
C4–H4A···O4 and C14–H14A···O3 hydrogen bonds (Table S1, Supplementary Materials) further connect
the dimers into a three-dimensional network (Figure 5).

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Figure 4. Intermolecular C–H···O hydrogen bonds (blue color) form dimeric structures in 4a.
Figure 5. Three-dimensional assembly in 4a with hydrogen bonds (blue dotted lines).
The asymmetric part of compound 4b consists of two independent molecules (A and B). Molecule A
adopts nearly-flat conformation whereas molecule B adopts nearly-perpendicular conformation (Table 1,
Figures 2 and 3). Molecule B is treated as whole-molecule disorder with 0.5:0.5 site occupancies ratio
in which the major and minor components are related with a two-fold rotation symmetry. In the crystal,
weak intermolecular C–H···O hydrogen bond links the molecules into one-dimensional infinite zigzag
chains along c-axis (Figure 6).

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Figure 6. Hydrogen-bonded infinite chain in 4b. Blue dotted lines are weak C–H···O
hydrogen bonds. The minor component of disordered molecule B is shown in pink.
Compounds 4c and 4d are two nearly-perpendicular conformers which show two-dimensional
structural similarity (Figure 7). In 4c, molecules are linked by weak C5–H5A···O1 hydrogen bond into
infinite chains, propagating along the crystallographic b-axis. In 4d, molecules are joined by three
weak intermolecular C4–H4A···O4, C5–H5A···O3 and C18–H18A···O5 hydrogen bonds (Table S1,
Supplementary Materials) into sheets parallel to (011) plane.
Figure 7. Two-dimensional structural similarities in (a) 4c and (b) 4d.
In compound 4e, the asymmetric unit consists of two independent molecules (A and B) and both
adopt nearly-planar conformation with maximum deviations of 0.343 Å and 0.275 Å from their respective
mean planes. The molecules are linked by weak C–H···O hydrogen bonds (Table S1, Supplementary
Materials) and π···π interactions into a three-dimensional network. The π···π interactions in 4e
(Table S2, Supplementary Materials) are the most redundant among all current compounds 4(a–e) due
to the layered structure. Additional information on C–H···π and π···π interactions can be found in
Supplementary Materials.
2.3. Antimicrobial Activities of the Synthesized Compound
The minimum inhibition concentration (MIC) values of compounds 4(a–e) against eight different
pathogenic microorganisms are given in Table 3. Compounds 4d with -methoxy and 4e with -nitro

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substitutions were found to inhibit all the tested microorganisms. Compound 4e gave the lowest
MIC value (125 µg/mL) against the Gram-negative bacterium Salmonella typhimurium ATCC 14028,
Gram-positive bacterium Streptococcus mutans ATCC 25175 and the yeast Candida albicans ATCC
10231. The MIC value of this compound against other bacteria was found to be 250 µg/mL. Compound
4d showed MIC value of 250 µg/mL against one Gram-negative bacterium (Salmonella typhimurium),
all the tested Gram-positive bacteria and the yeast, whereas MIC value of 500 µg/mL was observed
against the other Gram-negative bacteria. The MIC value for 4c was consistent (500 µg/mL) against all
tested microorganisms excluding Bacillus licheniformis. This bacterium was found to be resistant against
4c. No activity was found for 4b against all the tested microorganisms and this result may be due to the
-chloro substitution. In short, the results revealed the increase in antimicrobial activity follows with the
substitution of -methyl, -methoxy and -nitro, respectively, at the 4-position of the parental skeleton.
Table 3. Minimum inhibition concentration (MIC) of the synthesized compounds.
Gram Negative
Gram Positive
Yeast
Escherichia
coli
Klebsiella
pneumoniae
Pseudomonas
aeruginosa
Salmonella
Bacillus
Streptococcus Staphylococcus
Candida
albicans
Compound
typhimurium
licheniformis
mutans
aureus
ATCC 25922 ATCC 13883 ATCC 27853 ATCC 14028
ATCC 12759
ATCC 25175 ATCC 700699 ATCC 10231
4a
1000
-
1000
-
1000
-
1000
-
1000
-
1000
-
1000
-
1000
-
4b
4c
500
500
250
0.977
500
500
250
1.953
500
500
250
7.813
500
250
125
0.977
-
500
250
125
0.244
500
250
250
31.25
500
250
125
0.122
4d
4e
250
250
1.953
Tetracycline
2.4. Antioxidant Activities of the Synthesized Compound
The antioxidant capacities were systematically assessed using three different assays at an initial
concentration of 8 mg/mL (final concentration of 2 mg/mL). The scavenger capacity was determined
by measuring the decrease in absorption of diphenyl-2-picrylhydrazyl (DPPH) radicals. Meanwhile,
the reducing power was measured by ferric reducing antioxidant power (FRAP) method to observe the
reduction of ferric tripyridyltriazine (Fe(III)-TPTZ) complex to ferrous (Fe(II)-TPTZ) at low pH.
These two assays are mainly used to measure the direct involvement of the compounds in enhancing
the primary antioxidant activity, whereas the metal chelating assay measures the indirect ability of
compounds to act as secondary antioxidant by binding to ferrous (Fe(II)) ion catalyzing oxidation and
disrupting the formation of Fe(II)-ferrozine complex.
In general, low antioxidant activities were observed for the tested compounds which might be
related to the absence of hydroxyl moiety in the compounds. The results obtained from these assays
revealed the highest activity for compound 4e (32.62% ± 1.34%) in DPPH assay (Figure 8), compound
4d (31.01% ± 4.35%) in ferric reducing activity and compound 4a (27.11% ± 1.06%) in metal
chelating activity.

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Figure 8. DPPH scavenging, ferric reducing and metal chelating activities of compounds
4(a–e) at final concentration of 2 mg/mL. Each value represents the mean ± SD of
triplicate analyses.
3. Experimental Section
3.1. Instrumentation
The infrared spectra were recorded using a Perkin-Elmer System 2000 FTIR Spectrophotometer as
KBr disc in the frequency range of 4000–400 cm−1. The spectra for 1H-, 13C- and 1H-13C HMQC NMR
were recorded on a JEOL JNM-ECX 400 FT-NMR Spectrometer using deuterated CDCl3 as the solvent
and tetramethylsilane, TMS as the internal standard.
3.2. X-ray Diffraction Analysis
Bruker APEX II DUO CCD area-detector diffractometer was used to perform X-ray analysis on
these five colourless samples. MoKα radiation (λ = 0.71073 Å) was applied and φ and ω scans were
employed in the data collection. The raw data was first reduced using SAINT and absorption correction
process was carried out later by SADABS program. In this analysis, all the crystallographic data were
collected at room temperature. The SHELXTL [28] program was used to solve the structure with direct
methods. Refinements of the structures on F2 were done using full-matrix least-squares techniques.
Anisotropic refinements were applied on all non-hydrogen atoms. All C-bound hydrogen atoms were
calculated geometrically with the isotropic displacement parameters set to 1.2 (or 1.5 for methyl group)
times the equivalent isotropic U values of the parent carbon atoms. The Olex2 [29] software was used
to sketch the overlay diagrams. Crystallographic data for compounds 4(a–e) have been deposited
at the Cambridge Crystallographic Data Centre with CCDC deposition numbers of 1037756, 1037759
and 1037762-1037764 respectively. These data can be obtained free of charge via http://www.ccdc.
cam.ac.uk/conts/retrieving.html (or from the CCDC, 12 Union Road, Cambridge CB2 1EZ, UK; Fax:
+44 1223 336033; E-mail: deposit@ccdc.cam.ac.uk).

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3.3. Synthesis
3.3.1. Synthesis of 1-(Benzofuran-2-yl)ethanone (2) and -(Benzofuran-2-yl)-2-bromoethan-1-one (3)
Salicylaldehyde (1) (0.1 mol), chloroacetone (0.1 mol) and anhydrous potassium carbonate (30 g)
were dissolved in dry acetone (150 mL) and the mixture was refluxed for about 12 h. After the mixture
was cooled, the filtrate was removed under reduced pressure to obtain a yellow crude product of
1-(benzofuran-2-yl)ethanone (2) [30]. The crude product was recrystallized from petroleum ether and
purity of the product were checked by using TLC plate with silica gel and acetone:benzene (1:1) solvent
system. Next, 2 was refluxed with NBS and petroleum ether in methanol at 333 K for about 2 h. After
the reaction completed, the mixture was allowed to cool and filtered. The resultant crude product was
dried and recrystallized from ethanol to obtain 3 [25].
3.3.2. General Procedure for the Synthesis of 2‐(1‐Benzofuran‐2‐yl)‐2‐oxoethyl 4 (Unsubstituted/
substituted)benzoates 4(a–e)
1-(Benzofuran-2-yl)-2-bromoethan-1-one (3) (0.5 mol), substituted benzoic acid (0.6 mol) and
anhydrous potassium carbonate (0.5 g) were dissolved in dimethyl formamide (8 mL) and the mixture
was then stirred at room temperature for about 2 h. The progress of the reaction was monitored by TLC.
After the completion of the reaction, the mixture was poured into a 100 mL beaker containing crushed
ice. The precipitate formed was filtered, dried and recrystallized from acetone to get pure products of
4(a–e) (Figure 9) [25,31,32].
Figure 9. Reaction scheme for the synthesis of target compounds 4(a–e).
2-(1-Benzofuran-2-yl)-2-oxoethyl benzoate (4a): Solvent for crystal growth: Acetone; m.p.: 126–128 °C;
Yield: 78%; FTIR as KBr disc (cm−1): ν(C-H) aromatic 3124, 3062; ν(C-H) aliphatic 2953, ν(C=O)
1725, 1698; ν(C=C) 1559, 1475; ν(C-O) 1276, 1259. 1H-NMR (ppm) (CDCl3): δ: benzofuran protons
7.33 (t, 7.8 Hz, 1H); 7.48 (t, 7.8 Hz, 2H overlap with benzene proton); 7.52 (dd, 4.4 Hz, 1.4 Hz, 1H);
7.63 (s, 1H); 7.73 (d, 8.3 Hz, 1H); benzene protons 7.59 (d, 8.7 Hz, 2H); 8.15 (d, 8.2 Hz, 2H); CH2

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5.55 (s, 2H). 13C-NMR (ppm) (CDCl3): δ: benzofuran carbons 112.60, 113.59, 123.61, 124.30, 126.80,
128.84, 150.56, 155.74; benzene carbons 128.59, 129.29, 130.13, 133.58; CH2 66.36, COO 166.05,
C=O 183.84.
2‐(1‐Benzofuran‐2‐yl)‐2‐oxoethyl 4‐chlorobenzoate (4b): Solvent for crystal growth: Acetone; m.P.:
116–118 °C; Yield: 82%; FTIR as KBr disc (cm−1): ν(C-H) aromatic 3124, 3093, 3072; ν(C-H) aliphatic
1
2941, 2914; ν(C=O) 1723, 1697; ν(C=C) 1558, 1486; ν(C-O) 1271, ν(C-Cl) 1086. H-NMR (ppm)
(CDCl3): δ: benzofuran protons 7.34 (t, 7.8 Hz, 1H); 7.51 (td, 6.9 Hz, 0.9 Hz, 1H); 7.58 (d, 8.2 Hz,
1H); 7.63 (s, 1H); 7.72 (d, 7.8 Hz, 1H); benzene protons 7.44 (d, 8.7 Hz, 2H); 8.07 (d, 8.2 Hz, 2H);
13
CH2 5.54 (s, 2H). C-NMR (ppm) (CDCl3): δ: benzofuran carbons 112.60, 113.61, 123.63, 124.34,
127.75, 128.90, 150.49, 155.75; benzene carbons 126.77, 128.96, 131.51, 140.07; CH2 66.46, COO
165.22, C=O 183.59.
2‐(1‐Benzofuran‐2‐yl)‐2‐oxoethyl 4‐methylbenzoate (4c): Solvent for crystal growth: Acetone; m.p.:
154–156 °C; Yield: 79%; FTIR as KBr disc (cm−1): ν(C-H) aromatic 3115, 3098; ν(C-H) aliphatic 2932,
ν(C=O) 1721, 1682; ν(C=C) 1552, 1476; ν(C-O) 1269. 1H-NMR (ppm) (CDCl3): δ: benzofuran protons
7.33 (t, 7.3 Hz, 1H); 7.51 (t, 6.9 Hz, 0.9 Hz, 1H); 7.59 (d, 7.8 Hz, 1H); 7.62 (s, 1H); 7.72 (d, 7.8 Hz, 1H);
benzene protons 7.27 (d, 8.2 Hz, 2H); 8.03 (d, 8.2 Hz, 2H); CH3 2.42 (s, 3H); CH2 5.52 (s, 2H).
13C-NMR (ppm) (CDCl3): δ: benzofuran carbons 112.59, 113.54, 123.59, 124.27, 126.82, 128.79,
150.60, 155.74; benzene carbons 126.55, 129.31, 130.16, 144.34; CH3 21.85, CH2 66.24, COO 166.09,
C=O 183.99.
2‐(1‐Benzofuran‐2‐yl)‐2‐oxoethyl 4‐methoxybenzoate (4d): Solvent for crystal growth: Acetone; m.p.:
140–142 °C; Yield: 81%; FTIR as KBr disc (cm−1): ν(C-H) aromatic 3094, ν(C-H) aliphatic 2966, 2947;
ν(C=O) 1712, 1689; ν(C=C) 1549, 1475; ν(C-O) 1275, 1261. 1H-NMR (ppm) (CDCl3): δ: benzofuran
protons 7.32 (t, 7.8 Hz, 1H); 7.50 (t, 7.3 Hz, 1H); 7.58 (d, 8.7 Hz, 1H); 7.62 (s, 1H); 7.72 (d, 7.8 Hz,
1H); benzene protons 6.94 (d, 8.7 Hz, 2H); 8.09 (d, 8.7 Hz, 2H); OCH3 3.87 (s, 3H); CH2 5.51 (s, 2H).
13C-NMR (ppm) (CDCl3): δ: benzofuran carbons 112.59, 113.55, 123.59, 124.27, 126.82, 128.79,
150.59, 155.73; benzene carbons 113.85, 121.64, 132.23, 163.87; OCH3 55.58, CH2 66.15, COO
165.74, C=O 184.13.
2‐(1‐Benzofuran‐2‐yl)‐2‐oxoethyl 4‐nitrobenzoate(4e): Solvent for crystal growth: Acetone; m.p.:
180–182 °C; Yield: 84%; FTIR as KBr disc (cm−1): ν(C-H) aromatic 3116, ν(C-H) aliphatic 2924,
2858; ν(C=O) 1725, 1694; ν(C=C) 1610, 1475; ν(C-O) 1277, ν(NO2) 1524. 1H-NMR (ppm) (CDCl3):
δ: benzofuran protons 7.35 (t, 7.8 Hz, 1H); 7.53 (t, 7.4 Hz, 1H); 7.59 (d, 8.2 Hz, 1H); 7.65 (s, 1H); 7.74
(d, 7.8 Hz, 1H); benzene protons 8.32 (s, 4H); CH2 5.62 (s, 2H). 13C-NMR (ppm) (CDCl3): δ: benzofuran
carbons 112.59, 113.73, 123.67, 124.45, 126.74, 129.04, 150.91, 155.79; benzene carbons 123.75,
131.26, 134.72, 150.35; CH2 66.89, COO 164.23, C=O 183.03.
3.4. Antimicrobial Activities
Selected skin diseases microorganisms tested were obtained from the American Type Culture
Collection (ATCC). The Gram-positive bacteria used were Staphylococcus aureus ATCC 700699,

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Streptococcus mutans ATCC 25175, and Bacillus licheniformis ATCC 12759. Four Gram-negative
bacteria (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883, Pseudomonas aeruginosa
ATCC 27853 and Salmonella typhimurium ATCC 14028) and one yeast (Candida albicans ATCC 10231)
were also used for the test.
The minimum inhibition concentration (MIC) of each compound was determined using micro-well
dilution method as described by Sivasothy et al. [33]. The initial concentrations of each compound
ranged from 40–1.25 mg/mL. The lowest concentration which inhibited the growth of the respective
bacteria was considered as the MIC. Briefly, 5 µL of compound and 195 µL of bacteria inoculum were
added into each well of sterile 96-well plate (Nunc). The final inoculums size was approximately
1.5 × 106 CFU/mL for bacteria and yeast. The antibiotic tetracycline and DMSO (in similar volume
with tested compound) were respectively included as positive and negative controls in each plate. The
plates were then incubated at 37 °C for 20–22 h. An indicator of bacteria growth, 2-(4-iodophenyl)-3-
(4-nitrophenyl)-5-phenyltetrazolium chloride, 95% (INT) (Sigma Aldrich, St Louis, MO, USA) was
freshly prepared at initial concentration of 0.3 mg/mL. 40 µL of the solution was added to each well
and the plate was further incubated for another 30 min at 37 °C. The assay was performed in triplicate.
3.5. Antioxidant Activities
3.5.1. Diphenyl-2-picrylhydrazyl (DPPH) Radical Scavenging Assay
The free radical scavenging activity of each compound was estimated based on DPPH assay as
described by Ooi et al. [34], with slight modifications. Fifty microliters of the compound (with an initial
concentration of 8 mg/mL) was added to 150 µL of ethanolic DPPH solution (300 µM) while ethanol
was used as blank. For negative control, 50 µL of DMSO was added to the DPPH solution. The mixture was
left to stand for 30 min at 37 °C. Absorbance was read at 515 nm using Multiskan Spectrum microplate
reader (Thermo Scientific, Vantaa, Finland). The DPPH scavenging percentage was calculated as follows:
%the DPPH scavenging = [(absorbance of negative control − absorbance of sample)/
(1)
absorbance of negative control] × 100
All the experiments were performed in triplicate.
3.5.2. Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay of each compound was carried out based on method described by Ooi et al. [34].
The compound (50 μL) at initial concentration of 8 mg/mL was allowed to react with 150 μL of the
FRAP solution in a well of a 96-well plate. Triplicate of measurements were performed. The increase
in absorbance at 593 nm was measured using a Multiskan Spectrum microplate reader (Thermo Scientific)
after 20 min of incubation at 37 °C. DMSO was used as negative control while Trolox (Sigma Aldrich)
was used as positive control. The FRAP percentage was calculated as:
%inhibition = [absorbance of sample/maximum absorbance (3.8)] × 100
(2)

Molecules 2015, 20
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3.5.3. Metal Chelating (MC) Assay
The metal chelating assay was performed according to Ooi et al. [34]. Briefly, 50 µL of compound
(with an initial concentration of 8 mg/mL) was incubated with 5 µL ferrous chloride hexahydrate
(2 mM) and 130 µL of deionized water for 5 min. The reaction was initiated by the addition of 15 µL
of ferrozine (5 mM). DMSO was prepared as negative control and ethylenediaminetetraacetic acid
(EDTA) salt was prepared to be used as positive control. After the mixture has been incubated at room
temperature for 10 min, the absorbance was measured at 562 nm using Multiskan Spectrum microplate
reader (Thermo Scientific). The metal cheating percentage was calculated using the following equation:
%inhibition = [(absorbance of negative control − absorbance of sample)/absorbance of
(3)
negative control] × 100%
All experiments were performed in three replicates.
4. Conclusions
A series of five new 2‐(1‐benzofuran‐2‐yl)‐2‐oxoethyl 4-(un/substituted)benzoates 4(a–e) was
synthesized under mild conditions producing high purity with good yield. The synthesized products
4(a–e) are characterized by FTIR, 1H-, 13C- and 1H-13C HMQC NMR spectroscopic analysis and their
3D structures were confirmed by single-crystal X-ray diffraction studies. The X-ray diffraction studies
revealed the significance of various intermolecular H-bonding interactions contributing to the crystal
structure stability. Further, the compounds were screened for their antibacterial (four gram negative
and three gram positive bacterial strains) and antifungal (Candida albicans) activities using
tetracycline as standard. The results indicated the tested compounds possess antimicrobial ability
in the order of 4b < 4a < 4c < 4d < 4e. The highest potency with MIC value of 125 µg/mL was
observed for compound 4e. The results of antioxidant activities revealed the highest activity for
diphenyl-2-picrylhydrazyl (DPPH) radical scavenging, ferric reducing antioxidant power (FRAP)
assay and metal chelating (MC) activity are found in 4e (32.62% ± 1.34%), 4d (31.01% ± 4.35%)
and 4a (27.11% ± 1.06%), respectively. In conclusion, among the synthesized benzofuranyl esters,
compound 4e may be a potential antimicrobial and antioxidant agent. The results of the bioassay
revealed the vital effect of substituent change on antimicrobial and antioxidant activities.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/20/09/16566/s1.
Acknowledgments
CSCK thanks to Universiti Sains Malaysia (USM) for a postdoctoral research fellowship (2013–2015).
LYT thanks Malaysian Government for MyBrain15 (MyMaster) scholarship. CKQ, HKF, CSCK and TSC
thank Malaysian Government and USM for Research University Individual Grant (1001/PFIZIK/811278),
Fundamental Research Grant Scheme (FRGS) (203/PFIZIK/6711411) and Postgraduate Research Grant
Scheme (PRGS) (1001/PFIZIK/846076).The authors extend their appreciation to The Deanship of
Scientific Research at King Saud University for the research group project No. RGP VPP-207.

Molecules 2015, 20
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Author Contributions
C.S.C.K. conceived and designed the experiments; C.S.C.K. and L.Y.T. performed the experiments;
C.S.C.K., L.Y.T., T.S.C., S.C., Y.F.W., H.K.F. and C.K.Q. analyzed the data; S.F.S., N.S.H. and
K.L.O. performed the bioassays (antimicrobial and antioxidant); C.K.Q. contributed reagents/materials/
analysis tools; all authors wrote the paper; all authors read and approved the final manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
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© 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).