Synthesis of Kojic Ester Derivatives as Potential Antibacterial Agent

Article abstract of DOI:10.1155/2018/1245712

The search for lead product with beneficial pharmacological properties has become a great challenge and costly. Extraction and synthetic modification of bioactive compounds from natural resources has gained great attention and is cost effective. In this study, kojic acid was produced from fungal fermentation, using sago waste as substrate, and chemically incorporated with chalcones and azobenzene to form a series of kojic ester derivatives and evaluated for antibacterial activities. Kojic ester bearing halogenated chalcone demonstrated active inhibition against Staphylococcus aureus compared to that of standard ampicillin. The inhibition increased as the electronegativity of halogens decreased, while incorporation of azobenzene derivatives on kojic acid backbone demonstrated fair antibacterial activity against Escherichia coli with minimum inhibitory concentration (MIC) of 190-330 ppm. The presence of C=C and N=N reactive moieties in both chalcone and azo molecules contributed to the potential biological activities of the kojic acid ester.

Full text of DOI:10.1155/2018/1245712

Hindawi
Journal of Chemistry
Volume 2018, Article ID 1245712, 7 pages
https://doi.org/10.1155/2018/1245712
Research Article
Synthesis of Kojic Ester Derivatives as Potential
Antibacterial Agent
Carolynne Zie Wei Sie , Zainab Ngaini , Nurashikin Suhaili, and Eswaran Madiahlagan
Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia
Correspondence should be addressed to Zainab Ngaini; nzainab@unimas.my
Received 13 January 2018; Revised 9 March 2018; Accepted 19 March 2018; Published 6 May 2018
Academic Editor: Gabriel Navarrete-Vazquez
Copyright © 2018 Carolynne Zie Wei Sie et al. Tis is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly
cited.
Te search for lead product with benefcial pharmacological properties has become a great challenge and costly. Extraction and
synthetic modifcation of bioactive compounds from natural resources has gained great attention and is cost efective. In this study,
kojic acid was produced from fungal fermentation, using sago waste as substrate, and chemically incorporated with chalcones and
azobenzene to form a series of kojic ester derivatives and evaluated for antibacterial activities. Kojic ester bearing halogenated
chalcone demonstrated active inhibition against Staphylococcus aureus compared to that of standard ampicillin. Te inhibition
increased as the electronegativity of halogens decreased, while incorporation of azobenzene derivatives on kojic acid backbone
demonstrated fair antibacterial activity against Escherichia coli with minimum inhibitory concentration (MIC) of 190–330 ppm. Te
presence of C=C and N=N reactive moieties in both chalcone and azo molecules contributed to the potential biological activities
of the kojic acid ester.
1. Introduction
active compound which is derived from natural sources such
as fruits, vegetables, spices, tea, and soy-based foodstuf [17,
18] and used as an intermediate precursor of favonoids and
isofavonoids [19]. Chalcone is also commonly prepared via
Claisen Schmidt condensation. Te synthesis of chalcone
has drawn much attentions due to the presence of its ꢀ,ꢁ-
unsaturated ketone moieties which contributes to various
biological activities such as antimalaria [20], anticancer [21],
antiprotozoal, anti-infammatory, antibacterial, antiflarial,
anticonvulsant [22], antifungal, insect antifeedant, antimu-
tagenic [23], and antioxidant [24, 25]. Te presence of
halogen substituents particularly at the para position of chal-
cone backbone has been reported with excellent biological
activities such as antibacterial [26] and anti-infammatory
[27], while azobenzene bearing aryl/alkyl and N=N reactive
moieties have been extensively reported due to its excellent
antibacterial [28, 29], antioxidant [30], anti-infammatory
[31], antifungal [32], and antitumor [33] properties. Azoben-
zene is also recognized for its medicinal properties such as
antineoplastic [34], antidiabetic [35], and antiseptic [36].
Kojic acid is a natural pyrone produced from fungal fermen-
tation from various types of carbon substrates in the presence
of fungi [1, 2]. Kojic acid and its derivatives have drawn
attention and were studied intensively due to its signifcant
biological activities in medicine and pharmacological feld
such as antifungal, antibiotic [3, 4], anti-infammatory and
analgesic [5], and antibacterial [6] properties.
Many recent studies reported on chemical modifcation
of kojic acid for various applications such as antibacterial
activity [7] and dye sensitized solar cell (DSSC) [8]. Synthesis
of bioactive molecules employing kojic acid as a precursor
via esterifcation of available hydroxyl groups has been
widely reported to overcome the drawbacks of its hydrophilic
properties in various applications [9–12]. Esterifcation of
kojic acid, for instance, is able to enhance the hydrophobic
properties which in turn improved its performance as tyrosi-
nase inhibitor and antioxidant [13, 14].
Incorporation of kojic acid into other biological active
compounds has also been reported with enhanced biological
activities [15, 16]. Chalcones are an example of biological
Herein, we report on the isolation of kojic acid from
sago waste and utilized it as a precursor for the synthesis

2
Journal of Chemistry
of kojic acid ester bearing chalcone 3a–c and azobenzene
5a–d moieties. Tese compounds were screened for potential
antibacterial activities against E. coli and S. aureus, and the
58.0; H, 3.3%); ]max (KBr/cm−1) 3064 (C=CH), 1663 (C=O),
1
1607 (COO), 1583 (aromatic); H NMR (500 MHz, DMSO-
d ): ꢂ 8.13 (2H, d, ꢃ = 8.00 Hz, H-8), 8.05 (1H, d, ꢃ = 15.45 Hz,
6
efect of diferent moieties towards biological activities of
H-ꢁ), 8.02 (2H, d, ꢃ = 8.00 Hz, H-3), 7.98 (2H, d, ꢃ = 8.60 Hz,
H-9), 7.79 (1H, d, ꢃ = 15.45 Hz, H-ꢀ), 7.79 (2H, d, ꢃ = 8.00 Hz,
H-2).
kojic acid was studied.
2. Experimental
4-[(E)-3-(4-Chlorophenyl)-3-oxo-prop-1-enyl]benzoic Acid (2c).
Compound 2c (2.12 g, 74%) as a yellowish powder. m.p. 259-
260∘C; (Found: C, 67.09; H, 3.27%. C H ClO Requires C,
All the reagents were used as received without any further
purifcation. Melting points were determined in open capil-
lary tubes and are uncorrected. Te IR spectra were recorded
with KBr on Perkin Elmer 1605 FTIR spectrophotometer. Te
1H NMR were recorded in CDCl or DMSO-d on JEOL
16 11
3
67.0; H, 3.9%); ]max (KBr/cm−1) 2943 (C=CH), 1678 (C=O),
1
1612 (COO), 1577 (aromatic); H NMR (500 MHz, DMSO-
3
6
d ): ꢂ 8.20 (2H, d, ꢃ = 8.40 Hz, H-8), 8.00 (1H, d, ꢃ = 16.05 Hz,
6
ECA 500 spectrometer at 500 MHz using TMS as internal
standard. Te elemental carbon hydrogen nitrogen sulphur
(CHNS) elemental analyses were performed using Termo
Scientifc6 FLASH 2000 CHNS/O Analyzer.
H-ꢁ), 7.93 (2H, d, ꢃ = 8.40 Hz, H-3), 7.90 (2H, d, ꢃ = 8.40 Hz,
H-9), 7.78 (1H, d, ꢃ = 16.05 Hz, H-ꢀ), 7.65 (2H, d, ꢃ = 7.65 Hz,
H-2).
2.3. General Method for Synthesis of Kojic Acid-Chalcone
Derivatives (3a–c). Chalcone 2a–c (1.26 g, 0.05 mmol) was
added to a solution of 1 (0.71 g, 0.05 mmol), DMAP (0.12 g,
1 mmol), and DCC (1.03 g, 0.05 mmol) in DCM (30 mL). Te
reaction mixture was stirred at 0∘C for 4 h, washed with acetic
acid (1 M, 2 × 20 mL), followed by Na CO solution (1 M,
2.1. Fermentation of Kojic Acid (1). Sago waste was used as the
substrate for kojic acid fermentation. Prior to fermentation,
sago waste (5 g) was dried, ground, and sieved. Te medium
was supplemented with 3% (w/v) urea and 10% (w/v) mineral
salts solution containing KH PO , (w/v) MgSO ⋅7H O, and
2
4
4
2
2
3
yeast extract. Mixed strains of Aspergillus favus NSH9 and
A. favus Link 44-1 were added for fermentation process. Te
2 × 20 mL), and dried over magnesium sulphate. Te crude
product obtained was purifed by column chromatography
over silica gel (eluting with 1 : 2 EA/Hex) to aford title
compound.
∘
culture was incubated at 30 2 C in static condition for 20
days. Te slurry suspension culture (40 mL) was extracted
with ethyl acetate (2 × 15 mL). Te organic layer was evap-
orated in vacuo to form crude brown solid and recrystallized
from ethanol to aford 1 (0.15 g, 30%) as a yellowish needle
like solid; m.p. 149-150∘C [Reported 151–154∘C [37]]; ]max
(KBr/cm−1) 3173 (OH), 2919 (C=C), 1661 (C=O), 1228 (C-O);
[6-(Hydroxymethyl)-4-oxo-pyran-3-yl] 4-[(E)-3-oxo-3-phenyl-
prop-1-enyl] Benzoate (3a). Compound 3a (0.4 g, 23%) as a
white powder. m.p. 162-163∘C; (Found: C, 69.56; H, 4.25%.
C H O Requires C, 70.2; H, 4.3%); ]max (KBr/cm−1) 2987
1H NMR (500 MHz, DMSO-d ): ꢂ 9.04 (1H, s, OH-5), 8.02
22 16
6
6
(C=CH), 1730 (C=O ester), 1665 (C=O), 1644 (C=O), 1605
(s, 1H, H-6), 6.34 (1H, s, H-3), 5.67 (1H, s, OH-7), 4.29 (1H,
1
s, H-7); 13C NMR (125 MHz, DMSO-d ): ꢂ 174.4 (C-4), 168.6
(aromatic), 1201 (C-O); H NMR (500 MHz, DMSO-d ): ꢂ
6
6
8.69 (1H, s, H-6), 8.11 (7H, m, H-9, H-10, H-15, H-ꢁ), 7.79 (2H,
d, ꢃ = 16.05 Hz, H-ꢀ), 7.67 (1H, m, H-17), 7.59 (2H, m, H-16),
6.65 (1H, s, H-3), 5.02 (2H, s, H-7).
(C-2), 146.2 (C-5), 139.8 (C-6), 110.4 (C-3), 60.0 (C-7).
2.2. General Method for Synthesis of 4-Carboxyl Chal-
cone (2a–c). Acetophenone (1.2 g, 0.1 mmol) was added
to 4-carboxybenzaldehyde derivatives (0.15 g, 0.1 mmol) in
methanol (50 mL). KOH (1.7 g, 0.3 mmol) in methanol was
added and the mixture was stirred at room temperature
for 4 h under nitrogen atmosphere. Te precipitate formed
was fltered and recrystalized from ethanol to aford title
compound.
[6-(Hydroxymethyl)-4-oxo-pyran-3-yl]4-[(E)-3-(4-bromophe-
nyl)-3-oxo-prop-1-enyl] Benzoate (3b). Compound 3b (0.4 g,
19%) as a white powder. m.p. 194-195∘C; (Found: C,
58.37; H, 3.24%. C H BrO Requires C, 58.0; H, 3.3%);
22 15
6
]
(KBr/cm−1) 2926 (C=CH), 1697 (C=O ester), 1651
max
1
(C=O), 1623 (C=O), 1570 (aromatic), 1242 (C-O); H NMR
(500 MHz, DMSO-d ): ꢂ 8.67 (1H, s, H-6), 8.13 (2H, d, ꢃ =
6
8.6 Hz, H-15), 8.12 (2H, d, ꢃ 8.00 Hz, H-10), 8.04 (1H, d, ꢃ =
15.45 Hz, H-ꢁ), 7.99 (2H, d, ꢃ = 8.00 Hz, H-16), 8.00 (1H, d, ꢃ
= 12.6 Hz, H-ꢀ), 7.80 (2H, d, ꢃ = 8.55 Hz, H-9), 6.50 (1H, s,
H-3), 4.40 (2H, s, H-7).
4-[(E)-3-Oxo-3-phenyl-prop-1-enyl]benzoic Acid (2a). Com-
pound 2a (2.32 g, 92%) as a yellowish powder. m.p. 223-
224∘C; (Found: C, 76.61; H, 4.37%. C H O Requires C,
16 12
3
76.2; H, 4.8%); ]max (KBr/cm−1) 3059 (C=CH), 1677 (C=O),
1
1608 (COO), 1578 (aromatic); H NMR (500 MHz, DMSO-
d ): ꢂ 8.17 (2H, d, ꢃ = 7.45 Hz, H-3), 8.05 (1H, d, ꢃ = 16.05 Hz,
[6-(Hydroxymethyl)-4-oxo-pyran-3-yl]4-[(E)-3-(4-chloro-
phenyl)-3-oxo-prop-1-enyl]benzoate (3c). Compound 3c (0.5 g,
25%) as a white powder. m.p. 202-203∘C; (Found: C, 63.54;
H, 3.31%. C H ClO Requires C, 64.3; H, 3.7%); ]max
6
H-ꢁ), 8.02 (2H, d, ꢃ = 8.06 Hz, H-8), 7.99 (2H, d, ꢃ = 8.00 Hz,
H-2), 7.78 (1H, d, ꢃ = 15.45 Hz, H-ꢀ); 7.69 (1H, t, ꢃ = 7.73 Hz,
H-10), 7.59 (2H, t, ꢃ = 7.75 Hz, H-9).
22 15
6
(KBr/cm−1) 2927 (C=CH), 1731 (C=O ester), 1666 (C=O),
4-[(E)-3-(4-Bromophenyl)-3-oxo-prop-1-enyl]benzoic Acid (2b).
Compound 2b (2.65 g, 88%) as a yellowish powder; m.p. 246-
247∘C; (Found: C, 58.44; H, 3.65%. C H BrO Requires C,
1643 (C=O), 1607 (aromatic), 1239 (C-O); 1H NMR
(500 MHz, DMSO-d ): ꢂ 8.67 (1H, s, H-6), 8.13 (4H, t, ꢃ =
6
7.73 Hz, H-15, H-10), 8.04 (1H, d, ꢃ = 14.9 Hz, H-ꢁ), 7.99 (2H,
16 11
3

Journal of Chemistry
3
d, ꢃ = 8.0 Hz, H-16), 7.86 (1H, d, ꢃ = 11.45 Hz, H-ꢀ), 7.79 (2H,
d, ꢃ = 7.45 Hz, H-9), 6.50 (1H, s, H-3), 4.39 (2H, s, H-7).
the medium broth of inoculums added with DMSO with
no active compound. Te mixture was shaken at 230 rpm
at 37∘C. Te transmittances (T) of the aliquots were taken
every 1 h interval for six continuous hours using UV-Visible
Spectrophotometer Optima SP-300. Te antibacterial activity
was determined from the graph of In Nt versus time plotted.
Te In Nt values represent the number of colony forming
units (units/mL) which follows the equation of In Nt = 27.1
− 8.56 T [38, 39]
2.4. Synthesis of Azobenzene Derivatives (4a–d). HCl (8 M,
100 mL) was added drop wise to a solution of 4-aminoeth-
ylbenzoate (5 g, 30.0 mmol) in methanol (50 mL) in an ice
bath at 0–5∘C. Sodium nitrite (3.1 g, 45.0 mmol) in distilled
water (10 mL) and phenol (3.4 g, 36 mmol) in methanol
(50 mL) were added drop wise to the reaction mixture. NaOH
solution (1 M, 100 mL) was then added drop wise to pH
8.5–9.5. Te reaction mixture was stirred for 4 hr under
nitrogen atmosphere. Methanol (50 mL) was added and
acidifed with HCl (8 M, 50 mL) to form precipitate. Te solid
was fltered, washed, and recrystallized from ethanol. KOH
(0.13 g, 2.4 mmol) was added to the solid (0.2 g, 0.8 mmol)
in methanol (50 mL) and heated under refux for 4 h under
nitrogen atmosphere. Cold water (30 mL) was added to the
reaction mixture, acidifed with acetic acid (8 M), and fltered
and recrystallized from ethanol to give azobenzene interme-
diate. Azobenzene (1.5 g, 5 mmol) was added to a solution
of bromoalkene derivatives, t- BuOK (0.62 g, 5.0 mmol) and
KI (20 mg) in acetone (100 mL), and heated at refux for
48 h under nitrogen atmosphere. DCM (50 mL) and water
(30 mL) were added and the layers were separated. Te
aqueous layer was extracted with DCM (2 × 30 mL) and the
combined organic layers were dried and evaporated under
reduced pressure and recrystallized from ethanol to give title
compound 4a–d as colorless crystals. FTIR and NMR data
were consistent with the reported literature [8].
2.6.2. Via Kirby Bauer Disc Difusion Method. Te antibacte-
rial activities of the synthesized 2a–c and 3a–c were evaluated
against Escherichia coli and Staphylococcus aureus using Kirby
Bauer Disc Difusion method. E. coli and S. aureus were used
as inoculums and cultured in Mueller-Hinton Broth (MHB)
and incubated at 37.5∘C with constant shaking at 150 rpm for
18 h. Te bacteria suspension prepared was inoculated onto
the entire surface of a Mueller-Hinton Agar (MHA) plate with
sterilised cotton tipped swab to form an even lawn. Sterilised
flter paper disc impregnated with 10 ꢄL of the compound in
DMSO was placed on the bacteria surface of MHA plate using
a sterilised forcep. Te plates were incubated at 37.5∘C for 24 h.
Te zone of inhibition was measured in millimetres (mm) to
estimate the potency of the tested compound.
3. Results and Discussion
3.1. Preparation of Kojic Acid via Fermentation. Kojic acid
1 was prepared via fermentation of sago hampas employing
mixed strains of Aspergillus favus NSH9 and Aspergillus
favus Link 44-1. Aspergillus favus was used for a higher yield
of 1[15]. Te optimum parameters were from a combination
of 3% (w/v) urea and 10% mineral salts solution contain-
ing KH PO , (w/v) MgSO ⋅7H O and yeast. KH PO and
2.5. Synthesis of Kojic Acid-Azobenzene (5a–d). Tionyl chlo-
ride (0.036 g, 0.3 mmol) was added to a solution of 1 (0.014 g,
0.1 mmol) in DMF (20 mL) and heated under refux for 4 h.
Te reaction mixture was extracted with DCM (2 × 15 mL).
Te organic layer was dried, fltered, and concentrated under
reduced pressure to give kojyl chloride as yellowish needle
solid (0.012 g, 70%). Azo intermediate was added to a solution
of kojyl chloride prepared (0.1 g, 0.6 mmol) in DMF (30 mL)
with TEA (0.015 g, 0.15 mmol) in dried acetone (20 mL) and
heated under refuxed for 24 h. Te reaction mixture was
washed with dilute HCl (1 M, 2 × 20 mL). Te organic layer
was dried, fltered, and concentrated under reduced pressure.
Crude product was recrystallized from ethanol to aford 5b–e
as colorless crystals. FTIR and NMR data were consistent
with the reported literature [8].
2
4
4
2
2
4
MgSO supported the growth of bacteria strain and boosted
4
the production of kojic acid [40]. Yeast is a nitrogen source
for metabolic activation to enhance kojic acid production.
Its high levels of essential components such as vitamins and
oligoelements support growth of bacteria and fermentation
process [3].
3.2. Synthesis of Kojic Acid Ester Derivatives. Kojic ester 2a–c
was prepared via Claisen Schmidt condensation followed by
Steglich esterifcation with isolated kojic acid to yield 3a–c,
whereas kojic ester (5a–d) was prepared via kojyl chloride
[11] followed by addition of 4a–d to aford 5a–d (70–87%).
Te overall synthesis is shown in Scheme 1. Structures of
the compounds synthesized were confrmed by elemental
analysis, IR spectra, and 1H NMR spectra.
2.6. Antibacterial Screening
2.6.1. Turbidimetric Kinetic Method. Te antibacterial activ-
ities of synthesized 4a–d and 5a–d were screened against
Escherichia coli using turbidimetric kinetic method. Escher-
ichia coli were cultured on LB plate agar for 24 h at 37∘C
and a single colony was transferred to the media containing
nutrient broth prepared. Te inoculum was allowed to grow
at 37∘C with 250 rpm stirring overnight. Inoculum (0.2 mL)
was added to 10 mL of culture medium which had been added
with diferent concentrations (50 ppm, 80 ppm, and 100 ppm)
of respective compounds in DMSO. Tree replicates of
each concentration were prepared. Te control used was
Te IR spectra for 2a–c showed absorptions band at
3064–2943 cm−1 and 1678–1663 cm−1 attributed to C=C
and C=O group of chalcone linkage. A sharp band at
1612–1607 cm−1 indicated the presence of carboxyl group.
Absorption peaks at 1583–1577 cm−1 revealed the presence
1
of aromatic group. Te H NMR showed aromatic protons
of 2a at ꢂ 8.17–7.59 ppm as doublet and triplet while aro-
matic protons of 2b-c appeared at ꢂ 8.20–7.65 ppm as four
doublets with coupling constant 8–8.6 Hz. Te peaks of CH
ꢀ
= CH were observed at ꢂ 7.79–7.78 and 8.05–8.00 ppm as
ꢁ

4
Journal of Chemistry
X
O
O
O
O
O
OH
X
C（3
C（3
HO
X
O
O
O
KOH, MeOH
DCC, DMAP, DCM
HO
2a: X = H
2b: X = Br
2c: X = Cl
O
O
3a: X = H
3b: X = Br
3c: X = Cl
HO
O
O
HO
C（2
O
C（2
O
N
N（2
N
(1) SOCＦ2, DMF
O
(2) TEA, DMF
(i) HCl, NaN／3
(ii) ＃6（5OH, MeOH,
O
N
N
O
（3C
O
O
OH
(iii) C（2CHC（2Br tBuOK,
O
KI, acetone
4a: n = 2
4b: n = 3
4c: n = 5
4d: n = 6
5a: n = 2
5b: n = 3
5c: n = 5
5d: n = 6
HO
O
O
HO
HO
O
Scheme 1: Synthesis of kojic esters (3a–c) and (5a–d).
Table 1: MIC for 4a–d and 5a–d.
two doublets with ꢃ 15.5–16.1 Hz, which indicated trans
ab
confguration.
Compounds
MIC (ppm)
>400
>400
>400
>400
200
Formation of 3a–c was confrmed by FTIR and 1H NMR
spectroscopies. Te IR spectrum for 3a–c showed appear-
ance of C=O ester absorption band at 1731–1697 cm−1. Te
IR spectrum revealed two strong bands at 1666–1651 cm−1
and 1644–1623 cm−1 attributed to V(C=O) of chalcone
and kojic acid, respectively. Te absorption frequency at
1605–1570 cm−1 was attributed to aromatic group while ](C-
O) was represented by absorption band at 1242–1201 cm−1.
Te 1H NMR showed that all corresponding peaks indicated
the formation of 3a–c. Te existence of kojic acid moieties
in 3a–c was proven by appearance of two kojic acid olefnic
proton at ꢂ 8.69–8.67 and 6.65–6.50 ppm as singlet. Te
oxymethyl protons of kojic acid were represented by peaks
at ꢂ 5.02–4.39 ppm.
4a
4b
4c
4d
5a
5b
5c
5d
190
330
330
5a–d. Negative control, which is comprised of solvent and
inoculums, showed signifcant increase in growth of E. coli.
Te antibacterial assay was expressed by plotting graph of
In ꢅꢂ versus time. Te In ꢅꢂ value indicated number of colony
forming (growth of E. coli) units/mL which followed the
expression of In ꢅꢂ = 27.1 − 8.56 T [39].
Te NMR spectra of kojic ester (5a–d) via formation
of kojyl chloride revealed the presence of peak attributed
to oxymethyl proton (OCH ) at ꢂ 5.26–5.24 ppm as singlet,
2
which indicated 7-O-substituent 1 [41]. In comparison to 5-
O-substituted 3a–c, 7-O-substituted 5a–d showed shifed H-
6 olefnic proton signals to upfeld and oxymethyl proton
signals to downfeld. Carbonyl group of ketone is more
deshielded than carbonyl group of ester due to deshielding
efect of pyrone ring [42].
Kojic ester 4a–d exhibited weak inhibition while 5a–d
exhibited good inhibition against E. coli. Te inhibition of
5a–d is concentration dependent where 100 ppm > 80 ppm
> 50 ppm. Te minimum inhibitory concentration (MIC)
of 4a–d and 5a–d was determined by extrapolating the
concentration to zero growth rate of E. coli. Te MIC of 4a–d
and 5a–d is summarized in Table 1. Kojic ester moieties in
azobenzene are envisaged to increase the lipophilic properties
in 5a–d to interact and destruct the bacterial cell membrane
[43]. Te presence of C=O and N=N moieties in kojic ester
ofers more reactive sites that easily protonated under acidic
condition and reacted with the bacterial surfaces which
further enhance the biological activity [44]. Compound 5a-
b showed better activity than 5c-d due to the increase of
3.3. Antibacterial Activities of Kojic Ester Derivatives. Te
kojic ester derivatives 3a–c and 5a–d were evaluated for
antibacterial activities against gram negative strain E. coli
at 37∘C via turbidimetric method at three diferent con-
centrations, 50 ppm, 80 ppm, and 100 ppm, and compared
to chalcones (2a–c) and azobenzene (4a–d) derivatives.
Due to solubility limitation of 2a–c and 3a–d in organic
solvent, turbidimetric method was only applied on 4a–d and

Journal of Chemistry
Table 2: Inhibition zones of 2a–c and 3a–c.
5
of 2a–c increased with the decrease in electronegativity of
halogen substituent on chalcone with 2c showing the highest
inhibition, better than the ampicillin reference standard.
Lipophilic properties of kojic ester bearing chalcone deriva-
tives 3a-b inhibited better antibacterial activity. Kojic ester
5a–d bearing azobenzene derivatives showed good inhibition
against E. Coli compared to 4a–d. Te presence of C=O
ester, N=N, and alkene chains has increased the lipophilic
properties of of 5a–d and disrupts the bacteria cell walls.
Zone of inhibition (mm)
E. coli S. aureus
Compounds
2a
-
16.0
13.0
8.0
8.0
11.0
13.0
-
2b
-
2c
-
3a
-
3b
-
3c
-
-
-
Conflicts of Interest
DMSO
Kojic acid
Ampicillin
-
11.0
Te authors declare that there are no conficts of interest
16.0
regarding the publication of this paper.
Acknowledgments
chain length afer the cutof efect associated with decrease
in solubility.
Tis work was supported by Universiti Malaysia Sarawak and
Ministry of Education for Research Funds F07(DPP17)/1173/
2014(17) and FRGS/ST01(02)/968/2013(09). Te authors
would also like to express special thanks to Professor Dr. Ar-
bakariya Arif from UPM for providing the A. favus Link 44-
1 strain.
Antibacterial activity of kojic esters 2a–c and 3a–c bear-
ing chalcone moieties was conducted by measuring the zone
of inhibition on agar plates (Kirby Bauer Disc Difusion
method) against S. aureus and E. coli bacterial strain due
to solubility limitation in DMSO. Ampicillin was used as
reference standard and DMSO as negative control.
Te zone inhibition of 2a–c and 3a–c is shown in Table 2.
Kojic acid showed no inhibition against both S. aureus and E.
coli, while both series of 2a–c and 3a–c showed no inhibition
against E. coli compared to standard ampicillin. Compounds
2a–c and 3a–c, however, showed active inhibition against S.
aureus with 2c and 3a-b being inhibited better than ampi-
cillin. Te selective antibacterial activity of 2a–c and 3a–c
against gram positive bacterial strain is due to the signifcance
diferent in the membrane composition and architecture of
gram positive and gram negative organism [45]. S. aureus has
simpler mesh like cell membrane compared to E. coli which
eases the penetration of compound [46].
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