Synthesis, antioxidant and antimicrobial evaluation of simple aromatic esters of ferulic acid

Article abstract of DOI:10.1007/s12272-011-0803-y

Aromatic ester derivatives of ferulic acid where the phenolic hydroxyl is free (6a-d) or acetylated (5a-d) were evaluated for their antioxidant and antimicrobial properties. The superoxide radical scavenging capacity of compounds 5d and 6d-e (IC₅₀ of 0.19, 0.27 and 0.20 mM, respectively) was found to be twice as active as a-tocopherol (IC₅₀ = 0.51 mM). DPPH radical scavenging capacity was moderate and only found in compounds bearing free phenolic hydroxyl groups (6a-e). With regard to antimicrobial properties, compounds 6b and 6c displayed significant activity against Enterococcus faecalis (MICs = 16 μg/mL) and vancomycin-resistant E. faecalis (MIC for 6b, 32 and for 6c, 16 μg/mL). Compound 6c also demonstrated prominent activity against planktonic Staphylococcus aureus with a MIC value of <8 μg/mL and it inhibited bacterial biofilm formation by S. aureus with a MBEC value of <8 μg/mL, which was 64 and 128 times more potent than ofloxacin and vancomycin, respectively.

Full text of DOI:10.1007/s12272-011-0803-y

Arch Pharm Res Vol 34, No 8, 1251-1261, 2011
DOI 10.1007/s12272-011-0803-y
Synthesis, Antioxidant and Antimicrobial Evaluation of Simple
Aromatic Esters of Ferulic Acid
1
2
3
1
Burcu Çaliskan Ergün , Tülay Çoban , Fatma Kaynak Onurdag , and Erden Banoglu
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University, 06330 Etiler, Ankara, Turkey,
Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Ankara University, 06100 Tandogan, Ankara, Turkey, and
Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Gazi University, 06330 Etiler, Ankara, Turkey
2
3
(Received November 8, 2010/Revised February 12, 2011/Accepted March 13, 2011)
Aromatic ester derivatives of ferulic acid where the phenolic hydroxyl is free (6a-d) or acety-
lated (5a-d) were evaluated for their antioxidant and antimicrobial properties. The superoxide
radical scavenging capacity of compounds 5d and 6d-e (IC50 of 0.19, 0.27 and 0.20 mM, respec-
tively) was found to be twice as active as
α-tocopherol (IC50 = 0.51 mM). DPPH radical scav-
enging capacity was moderate and only found in compounds bearing free phenolic hydroxyl
groups (6a-e). With regard to antimicrobial properties, compounds 6b and 6c displayed signif-
icant activity against Enterococcus faecalis (MICs = 16
faecalis (MIC for 6b, 32 and for 6c, 16
activity against planktonic Staphylococcus aureus with a MIC value of <8
µ
g/mL) and vancomycin-resistant E.
g/mL). Compound 6c also demonstrated prominent
g/mL and it inhib-
g/mL, which was 64
µ
µ
ited bacterial biofilm formation by S. aureus with a MBEC value of <8
and 128 times more potent than ofloxacin and vancomycin, respectively.
µ
Key words: Ferulic acid, DPPH, Superoxide, Antimicrobial, Biofilm
INTRODUCTION
venging activities bearing potential for treatment of
inflammation and cancer (Murakami et al., 2002;
Ferulic acid (FA) (3), 3-(4-hydroxy-3-methoxyphenyl)- Nenadis et al., 2003; Jayaprakasam et al., 2006).
2-propenoic acid, acts as an effective radical scavenger These types of lipophilic FA derivatives have also
due to its ability to form a resonance stabilized phen- been shown to inhibit phorbol ester-induced Epstein-
oxy radical with its phenolic nucleus and its extended Barr virus activation and superoxide anion generation
side-chain conjugation (Graf, 1992). The various ester in cellular systems (Murakami et al., 2000). Moreover,
forms of FA are known to behave as potent antioxid- FA is reported to have antimicrobial activity against
ants in plants and plant-derived foods by trapping Gram-positive, Gram-negative bacteria and yeasts
and stabilizing radical species (Masuda et al., 2006). (Jeong et al., 2000) in which the antimicrobial mecha-
Since researchers has been able to produce FA in mass nism of FA was attributed to its inhibition of arylamine
quantities from rice bran, the utilization of this dietary
N-acetyltransferase in the bacteria (Lo and Chung,
phenolic compound for synthetic approaches has become 1999). In support of these findings, the antimicrobial
very attractive to develop FA-derived novel chemopre- activity of the synthetic alkyl ferulates (Michiyo et al.,
ventive strategies as antioxidant and anticancer agents 2002; Ou and Kwok, 2004) and some phenolic plant
(
Mori et al., 1999; Taniguchi et al., 1999). For example, extracts containing FA have also been reported (Panizzi
different alkyl esters of FA have recently emerged as et al., 2002; Proestos et al., 2005). In addition, gerany-
synthetic agents with antioxidant and radical sca- lation of the phenolic group in FA was shown to in-
hibit bacterial biofilm formation by Porphyromonas
gingivalis and Streptococcus mutants (Bodet et al.,
Correspondence to: Erden Banoglu, Department of Pharmaceu-
2008). Considering the promising results with alkyl
tical Chemistry, Faculty of Pharmacy, Gazi University, Taç Sk.
6330 Etiler, Ankara, Turkey
ferulates, we now prepared simple aromatic ester de-
rivatives of FA (Table I) and assessed their anti-
oxidant potential and antimicrobial activities.
0
Tel: 90-312-202-3236, Fax: 90-312-223-5018
E-mail: banoglu@gazi.edu.tr
1251

1252
B. Ç. Ergün et al.
®
Table I. Synthesized aromatic esters of ferulic acid
tography was performed with a Combiflash Rf auto-
mated flash chromatography system (Teledyne-Isco)
using hexane-ethyl acetate or dichloromethane-metha-
nol solvent gradients. Final purity of the compounds
before biological testing was determined by ultra pres-
sure liquid chromatography (UPLC) with UV detection
using acetonitrile/water solvent gradient.
Compound
FA
R
Ar
3
(
)
H
Ac
H
H
4
5
a
Ac
H
Synthesis of FA (3)
FA was synthesized by Knoevenagel condensation
between vanillin and malonic acid as previously re-
6a
o
o
ported (Xia et al., 2008). M.p. 171-172 C; lit. 172-173 C
5
b
b
Ac
H
−1
(
Xia et al., 2008). IR (KBr, cm ): 3440 (O-H), 1696
C=O). HRMS C H O (M-1): calcd. 193.0501;
found: 193.0503.
(
m/z
6
10 10
4
5
c
c
Ac
H
Synthesis of O-acetyl FA (4)
-acetyl FA was prepared by the reaction of FA
with acetic anhydride in pyridine using DMAP as
O
o
o
6
a catalyst. M.p. 199-202 C; lit. 199-200 C (Galey and
−1
Terranova, 1996). IR (KBr, cm ): 3274-2037 (broad,
COOH), 1770 (C=O), 1690 (C=O). HRMS C H O
5
12
12
5
d
d
Ac
H
(M+1): calcd. 237.0763; found: 237.0764.
6
General synthesis of esters of O-acetyl FA (5a-e)
To a mixture of -acetyl FA (1 mmol) in dry di-
chloromethane (DCM; 5 mL), oxalyl chloride (2 mmol)
and DMF (25 L) were added, and the resulting solu-
5
e
e
Ac
H
O
6
µ
tion was stirred under argon atmosphere for 15 min at
rt. After evaporation of the solvent, the residue was
dissolved in dry DCM (5 mL) and the appropriate
phenol derivative (1 mmol) and TEA (2.5 mmol) were
MATERIALS AND METHODS
Vanillin, malonic acid, hydrazine hydrate, dimethyl- added. The reaction mixture was stirred for 45 min at
aminopyridine (DMAP), triethylamine (TEA), oxalyl rt under argon atmosphere. After addition of ice-
chloride, aniline, acetic anhydride, pyridine and phenol water, the pH of the medium was adjusted to 4-5 with
derivatives were purchased from Aldrich Chemical acetic acid. The separated organic phase was washed
and Merck Chemical. Xanthine, xanthine oxidase, with water, dried over anhydrous Na
cytochrome c, 2,2-diphenyl-1-picrylhydrazyl (DPPH), solvent was evaporated under vacuum. The residue
butylated hydroxytoluene, -tocopherol, thiobarbituric was purified by automated flash column chromatog-
acid (TBA) were purchased from Sigma Chemical. IR raphy using hexane-EtOAc or DCM-MeOH as eluents.
KBr) spectra were recorded on a Mattson 1000 FTIR
2 4
SO and the
α
(
1
spectrometer. H-NMR spectra were recorded in DMSO- 4-Tert-butylphenyl 3-(4-acetyloxy-3-methoxyphen-
on a Varian Mercury 400, 400 MHz High Perfor- yl)-2-propenoate (5a)
mance Digital FT-NMR spectrometer using tetrame- Elution with hexane-EtOAc 0-25% afforded 5a as a
d
6
o
−1
thylsilane as the internal standard at the NMR faci- white solid (yield 74%); m.p. 156-157 C; IR (KBr, cm ):
1
lity of the Faculty of Pharmacy, Ankara University. 1776 (C=O), 1728 (C=O); H-NMR (DMSO-d
All chemical shifts were recorded as (ppm). High re- (s, 9H, (CH ) ), 2,27 (s, 3H, COCH ), 3.84 (s, 3H, OCH ),
solution mass spectra (HRMS) were performed on a 6.96 (d, J = 16 Hz, 1H,
6
): δ 1.30
δ
3
3
3
3
α
-H), 7.13 (d, J = 8.8 Hz, 2H,
Waters LCT Premier XE orthogonal acceleration time- ArH), 7.17 (d, J = 8.4 Hz, 1H, ArH), 7.39 (dd, J = 8.4,
of-flight (oa-TOF) mass spectrometer with using ESI 1.6 Hz, 1H, ArH), 7.45 (d, J = 8.8 Hz, 2H, ArH), 7.62
(
+) or ESI (
−
) methods (Waters Corporation). Melting (d, J = 1.6 Hz, 1H, ArH), 7.84 (d, J = 16 Hz, 1H,
β
-H);
points were determined with an SMP-II Digital Melting HRMS for C22
Point Apparatus and are uncorrected. Flash chroma- 369.1709.
24 5
H O (M+1): calcd. 369.1702; found:

Biological Activity of Ferulic Acid Derivatives
1253
4-Iso-propylphenyl 3-(4-acetyloxy-3-methoxyphen- derivative (5a-e) (0.5 mmol) in acetonitrile (5 mL) was
yl)-2-propenoate (5b)
added hydrazine monohydrate (1.5 mmol), and stirred
Elution with hexane-EtOAc 0-25% afforded 5b as a for 15 min at RT. After addition of acetic acid and
o
−1
white solid (yield 77%); m.p. 132-133 C; IR (KBr, cm ): water to the reaction mixture, the precipitate formed
1
1
(
770 (C=O), 1728 (C=O); H-NMR (DMSO-d
d, J = 6.8 Hz, 6H, (CH ), 2.25 (s, 3H, COCH
(CH ) ), 3.82 (s, 3H, OCH ), 6.93 (d, J = 16
6
):
3
δ
1.19 was purified by recrystallization from the appropriate
), 2.90 solvent.
3 2
)
(
m, 1H, C
H
3
2
3
Hz, 1H,
α
-H), 7.09 (m, 2H, ArH), 7.14 (d, J = 8.0 Hz, 4-Tert-butylphenyl 3-(4-hydroxy-3-methoxyphen-
1H, ArH), 7.28 (m, 2H, ArH), 7.36 (dd, J = 8.4, 2.0 Hz, yl)-2-propenoate (6a)
1
H, ArH), 7.60 (d, J = 2 Hz, 1H, ArH), 7.81 (d, J = 16 Crystallization from MeOH-water afforded 6a as a
o
−1
Hz, 1H,
β-H); HRMS for C H O (M+1): calcd. white solid (yield 83%); m.p. 160-161 C; IR (KBr, cm ):
21 22 5
1
355.1545; found: 355.1535.
3557 (O-H), 1722 (C=O); H-NMR (DMSO-d
6
): δ 1.30
(
s, 9H, (CH ) ), 3.83 (s, 3H, OCH ), 6.72 (d, J = 16 Hz,
3
3
3
(
4-chloro-3-methyl)Phenyl 3-(4-acetyloxy-3-meth- 1H,
oxyphenyl)-2-propenoate (5c) Hz, 2H, ArH), 7.20 (dd, J = 8.4, 1.6 Hz, 1H, ArH), 7.43
Elution with hexane-EtOAc 0-25% afforded 5c −as (m, 3H, ArH), 7.74 (d, J = 16 Hz, 1H, -H); HRMS for
white solid (yield 70%); m.p. 122-123 C; IR (KBr, cm ): C H O (M+1): calcd. 327.1596; found: 327.1586.
α-H), 6.82 (d, J = 8.0 Hz, 1H, ArH), 7.09 (d, J = 8.8
β
o
1
20
22
4
1
1
6
765 (C=O), 1728 (C=O); H-NMR (DMSO-d ): δ 2.25
(s, 3H, COCH ), 2.32 (s, 3H, CH ), 3.82 (s, 3H, OCH ), 4-Iso-propylphenyl 3-(4-hydroxy-3-methoxyphen-
3
3
3
6
1
2
7
.93 (d, J = 16 Hz, 1H, α-H), 7.08 (dd, J = 8.8, 2.8 Hz, yl)-2-propenoate (6b)
H, ArH), 7.15 (d, J = 8.0 Hz, 1H, ArH), 7.24 (d, J = Crystallization from methanol-water mixture afforded
o
.8 Hz, 1H, ArH), 7.37 (dd, J = 8.4, 1.6 Hz, 1H, ArH), 6b as a white solid (yield 55%); m.p. 134-135 C; IR
−1
1
.46 (d, J = 8.8 Hz, 1H, ArH), 7.60 (d, J = 1.6 Hz, 1H, (KBr, cm ): 3509 (O-H), 1722 (C=O); H-NMR (DMSO-
ArH), 7.83 (d, J = 15.6 Hz, 1H,
β
-H); HRMS for
d
C
6
):
H
δ
1.21 (d, J = 6.4 Hz, 6H, (CH
3 2
) ), 2.92 (m, 1H,
C H ClO (M+1): calcd. 361.0843; found: 361.0836.
(CH ) ), 3.83 (s, 3H, OCH ), 6.71 (d, J = 15.6 Hz,
19
17
5
3 2
3
1
H,
-Methoxyphenyl 3-(4-acetyloxy-3-methoxyphen- 8.8 Hz, 2H, ArH), 7.20 (dd, J = 8.4, 1.6 Hz, 1H, ArH),
yl)-2-propenoate (5d) 7.29 (d, J = 8.8 Hz, 2H, ArH), 7.42 (d, J = 1.6 Hz, 1H,
Elution with DCM-MeOH 0-2% afforded 5d as a white ArH), 7.73 (d, J = 15.6 Hz, 1H, -H), 9.70 (bs, 1H, OH);
(M+1): calcd. 313.1440; found:
α-H), 6.82 (d, J = 7.6 Hz, 1H, ArH), 7.09 (d, J =
4
β
o
−1
20 4
solid (yield 60%); m.p. 151-152 C; IR (KBr, cm ): 1770 HRMS for C19H O
1
(C=O), 1744 (C=O); H-NMR (DMSO-d
6
): δ 2.27 (s, 3H, 313.1434.
COCH ), 3.77 (s, 3H, OCH ), 3.84 (s, 3H, OCH ), 6.93
3
3
3
(
2
d, J = 16 Hz, 1H, α-H), 6.98 (m, 2H, ArH), 7.13 (m, (4-chloro-3-methyl)Phenyl 3-(4-hydroxy-3-meth-
H, ArH), 7.17 (d, J = 8.4 Hz, 1H, ArH), 7.38 (dd, J = oxyphenyl)-2-propenoate (6c)
8
.4, 2 Hz, 1H, ArH), 7.62 (d, J = 1.6 Hz, 1H, ArH), 7.83 Crystallization from MeOH-water afforded 6c as a
o
−1
(d, J = 16 Hz, 1H,
18 6
β-H); HRMS for C19H O (M+1): beige solid (yield 60%); m.p. 113-114 C; IR (KBr, cm ):
1
calcd. 343.1182; found: 343.1181.
3434 (O-H), 1717 (C=O); H-NMR (DMSO-d
6
): δ 2.34
(
s, 3H, CH ), 3.83 (s, 3H, OCH ), 6.71 (d, J = 16 Hz,
3
3
4
-(acetylamino)Phenyl 3-(4-acetyloxy-3-methoxy- 1H,
phenyl)-2-propenoate (5e) 8.4, 2.8 Hz, 1H, ArH), 7.20-7.23 (m, 2H, ArH), 7.42 (d,
Elution with DCM-MeOH 0-5% afforded 5e as beige J = 2.0 Hz, 1H, ArH), 7.47 (d, J = 8.8 Hz, 1H, ArH),
α-H), 6.82 (d, J = 8.0 Hz, 1H, ArH), 7.07 (dd, J =
o
o
solid (yield 70%); m.p. 234-235 C; lit. 246-250 C (Del 7.75 (d, J = 16 Hz, 1H,
Soldato et al., 2002). IR (KBr, cm ): 3376 (N-H), 1744 for C17H15ClO (M+1): calcd. 319.0737; found: 319.0739.
β-H), 9.75 (bs, 1H, OH); HRMS
−
1
4
1
(C=O), 1722 (C=O), 1685 (C=O); H-NMR (DMSO-d ):
6
δ
2.05 (s, 3H, COCH
3
), 2.27 (s, 3H, COCH
3
), 3.84 (s, 4-Methoxyphenyl 3-(4-hydroxy-3-methoxyphen-
3
H, OCH ), 6.94 (d, J = 16 Hz, 1H, α
3
-H), 7.14 (m, 2H, yl)-2-propenoate (6d)
ArH), 7.17 (d, J = 8.0 Hz, 1H, ArH), 7.38 (dd, J = 8.4, 6d was obtained as a white solid (yield 88%); m.p.
o
−1
1
1
3
.6 Hz, 1H, ArH), 7.61-7.62 (m, 3H, ArH), 7.84 (d, J = 146-147 C; IR (KBr, cm ): 3418 (O-H), 1733 (C=O);
1
6 Hz, 1H,
70.1291; found: 370.1277.
β
-H); HRMS for C20
H O
19 6
(M+1): calcd.
H-NMR (DMSO-d
6
):
δ
3.76 (s, 3H, OCH
3
), 3.83 (s, 3H,
OCH ), 6.69 (d, J = 15.6 Hz, 1H,
Hz, 1H, ArH), 6.97 (m, 2H, ArH), 7.10 (m, 2H, ArH),
α-H), 6.82 (d, J = 8.0
3
Synthesis of esters of FA (6a-e)
To a solution of the appropriate
7.20 (dd, J = 8.0, 2.0 Hz, 1H, ArH), 7.41 (d, J = 2.0 Hz,
-acetyl FA ester 1H, ArH), 7.73 (d, J = 16 Hz, 1H, β-H); HRMS for
O

1254
B. Ç. Ergün et al.
C H O (M+1): calcd. 301.1076; found: 301.1078.
pressed as the inhibition percentage and were cal-
culated using the formula: radical scavenging activity
17
16
5
4
-(acetylamino)Phenyl 3-(4-hydroxy-3-methoxy- (%) = [(A
phenyl)-2-propenoate (6e) for control (blank, without compound) and A is the
Crystallization from MeOH-water afforded 6e as beige absorbance in the presence of compound.
0
1 0 0
− A /A ) × 100], where A is the absorbance
1
o
o
solid (yield 72%); m.p. 194-197 C; lit. 185-195 C (Del
Soldato et al., 2002). IR (KBr, cm ): 3445-3200 (N-H, Lipid peroxidation (LP) assay
O-H), 1733 (C=O), 1674 (C=O); H-NMR (DMSO-d
−1
1
6
):
), 6.67 (d, J = (1980). Procedures involving the animals and their
-H), 6.79 (d, J = 8.4 Hz, 1H, ArH), 7.08 care conformed to institutional guidelines in compli-
δ
LP was assessed by the method of Mihara et al.
2.02 (s, 3H, COCH
3 3
), 3.81 (s, 3H, OCH
15.6 Hz, 1H,
α
(
d, J = 9.2 Hz, 2H, ArH), 7.18 (dd, J = 8.0, 2.0 Hz, 1H, ance with national and international laws and guide-
ArH), 7.39 (d, J = 1.6 Hz, 1H, ArH), 7.58 (d, J = 9.2 lines for the use of animals in biomedical research.
Hz, 2H, ArH), 7.71 (d, J = 15.6 Hz, 1H, -H), 9.68 (bs, Animals were fasted 24 h prior to sacrifice and then
H, OH), 9.98 (bs, 1H, NH); HRMS for C H NO
β
1
sacrificed by decapitation under anesthesia. The livers
were immediately removed and washed in ice-cold
distilled water, then immediately homogenized with a
Teflon homogenizer. LP was measured spectrophoto-
18
17
5
(M+1): calcd. 328.1185; found: 328.1189.
Antioxidant activity
Superoxide anion radical (O
tivity
•−) scavenging ac- metrically by estimation of thiobarbituric acid react-
2
ants (TBARS). Amounts of TBARS were expressed in
The capacity of FA derivatives to scavenge O 2• − was terms of nmol malondialdehyde (MDA/g tissue). A
determined spectrophotometrically on the basis of typical optimized assay mixture contained 0.5 mL of
inhibition of cytochrome c reduction according to the liver homogenate, 0.1 mL of Tris-HCl buffer (pH 7.2),
modified method of McCord and Fridovich (McCord 0.05 mL of 0.1 mM ascorbic acid, 0.05 mL of 4 mM
•
−
and Fridovich, 1969). O
2
was generated by a xanthine- FeCl
2
and 0.05 mL of various concentrations of FA
-tocopherol; this was incubated for 1 h
mL, total volume) consisted of phosphate buffer (pH at 37 C. After incubation, 3.0 mL of H PO and 1 mL
xanthine oxidase system. The incubation mixture (1 derivatives or
α
o
3
4
7
(
.8, 0.05 M), xanthine oxidase (0.32 Units/mL), xanthine of 0.6% TBA were added and shaken vigorously. The
50 M), cytochrome c (60 mM) and different concen- mixture was boiled for 30 min. After cooling the
trations of FA derivatives at 100 L. The reaction was mixture to room temperature, n-butanol was added
µ
µ
initiated by the addition of xanthine oxidase to this and vigorously mixed. The n-butanol phase was sep-
mixture. The absorbance was measured at 550 nm for arated at 3000 rpm for 10 min. The absorbance of the
3
min (cytochrome c reduction). Each experiment was supernatant was read at 532 nm against a blank,
performed in triplicate and the results are expressed which contained all reagents except the liver homo-
as a percent of the control (DMSO).
genate.
DPPH radical scavenging assay
DPPH assays were performed using purified sam-
Microbiology
Escherichia coli ATCC (American Type Culture
ples of test compounds as previously described (Blois, Collection) 25922, Klebsiella pneumoniae RSKK
958). Test samples were dissolved in DMSO and (Refik Saydam Culture Collection) 574, Staphyloco-
mixed with methanol solutions of DPPH (100 mM) in ccus aureus ATCC 29213, E. faecalis ATCC 29212,
1
9
3
5
6-well micro titer plates, followed by incubation at Candida albicans ATCC 10231, C. krusei ATCC 6258,
7 C for 30 min. DPPH reduction was estimated at C. pa- rapsilosis ATCC 90028 and clinical isolates of
17 nm. For each test compound, different concentra- the bacteria were included in the study. Clinical
o
tions were tested. Final concentrations of test materials isolates of E. coli and K. pneumoniae had extended
were typically in the range from 0.0675 to 1 mM. Per- spectrum beta lactamase (ESBL) and methicilline
cent inhibition by sample treatment was determined resistant S. aureus isolates (MRSA) were resistant to
by comparison with a DMSO-treated control group. all beta-lactam antibiotics. E. faecalis isolates were
All experiments were carried out in triplicate. The resistant to vancomycin (VRE).
antioxidant activity of each test compound was ex-
Standard powders of ampicillin (Mustafa Nevzat
pressed as an IC50 value ± S.D. which was calculated Pharma), gentamicin sulphate (Paninkret Chem.
by linear regression analysis. FA was used as a Pharm.), ofloxacin (Zhejiang Huangyan East Asia
positive control and its IC50 value was found to be 0.1 Chemical Co. Ltd.), meropenem (Astra Zeneca), van-
±
0.04 µM. The radical scavenging activities were ex- comycin (Mayne Pharma), fluconazole (Sigma), keto-

Biological Activity of Ferulic Acid Derivatives
1255
conazole (Sigma), itraconazole (Sigma), 5-fluorocytosin ofloxacin, vancomycin and amphotericin-B and the
Sigma) and amphotericin B trihydrate (Riedel de tested compounds against biofilm forms of the strains.
Haen) were dissolved in appropriate solvents recom- Subcultures of S. aureus and E. faecalis were grown
mended by Clinical and Laboratory Standards Institute on Tryptic Soy Agar (TSA; Merck) for 24 h at 37 C and
(
o
(
CLSI) guidelines (Rex et al., 2008). Stock solutions of subcultures of the Candida strains were grown on
o
the tested compounds were dissolved in DMSO. Stock SDA for 24-48 h at 35 C. After checking the purity
solutions of the tested compounds and standard drugs and viability of the cultures, a second subculture was
were diluted two-fold in microplate wells. All solvents grown on TSA and SDA plates for bacteria and fungi,
and diluents, pure microorganisms and pure media respectively and used for the susceptibility testing.
were used in control wells. All the experiments were McFarland-1 standard culture suspension was pre-
done in 3 parallel series.
Bacteria were subcultured in Mueller Hinton Agar used as the inoculum in the assay. Twenty-two milli-
MHA) (Merck) plates and incubated overnight at liters of the inoculum was poured to the trough of the
pared and a 30-fold dilution of this suspension was
(
3
o
7 C and Candida were subcultured in Sabouraud MBEC Plate and then the peg lid was placed onto the
o
Dextrose Agar (SDA; Merck) plates at 35 C for 24-48 trough. The device was placed on a rotary shaker,
o
h.
which was set to 10 of inclination with 3-6 rocks per
Bacterial susceptibility testing was performed min. Biofilms were formed on the lid of the device
o
o
according to the guidelines of CLSI M100-S18 (Wikler during incubation for 24 h at 37 C and 48 hat 35 C for
et al., 2009). Mueller Hinton Broth (MHB; Merck) was bacteria and fungi, respectively. At the same condi-
added to each microplate well. The bacterial suspen- tions and inoculum, a modified microtitre plate test
5
sions used for inoculation were prepared at 10 CFU/ was applied to all strains and biofilm formation was
mL by diluting fresh cultures at McFarland 0.5 dens- confirmed (Abdi-Ali et al., 2006). After incubation, the
7
5
ity (10 CFU/mL). Suspensions of the bacteria at 10
CFU/mL concentrations were inoculated with a two- dilutions of the antibacterial agents and the compounds
fold diluted solution of the compounds. A 10 L bac- were prepared in tryptic soy broth (TSB; Merck) and
peg lid was transferred to a 96-well plate in which
µ
teria inoculum was added to each microplate well. dilutions of amphothericin-B and the compounds were
There were 10 CFU/mL bacteria in the wells after in- prepared in RPMI-1640 medium with L-glutamine
oculations. Microplates were incubated at 37 C over- buffered to pH 7 with MOPS. These plates were also
4
o
night. After incubation, the lowest concentration of incubated for the appropriate conditions for bacteria
the compounds that completely inhibited macroscopic and fungi. After incubation, the peg lid was rinsed
growth was determined and reported as the minimum twice in physiological saline and transferred to another
inhibitory concentration (MIC).
microplate containing neutralizing agents and the
Fungal susceptibility testing was performed accord- recommended recovery medium. The biofilms were
ing to the guidelines of CLSI M27-A3 (Rex et al., then disrupted into this medium by sonication. After
2008). Roswell Park Memorial Institute (RPMI)-1640 sonication, the peg lid was removed from the recovery
medium with L-glutamine (Sigma) buffered to pH 7 medium plate and after incubation, lowest concentra-
with 3-(N-morpholino)propanesulfonic acid (MOPS) tion of the agents and the compounds that completely
(Sigma) was added each microplate well. The colonies inhibited macroscopic growth was determined as the
were suspended in sterile saline and the resulting MBECs.
suspension was adjusted to McFarland 0.5 density
6
(
10 CFU/mL). A working suspension was prepared by RESULTS
a 1:100 dilution followed by a 1:20 dilution of the stock
3
suspension. A 10
µ
L of this suspension at 10 CFU/mL Chemistry
FA ( ) was synthesized by reaction of vanillin (
compounds. Microplates were incubated at 35 C for with malonic acid ( ) by a Knoevenagel-type conden-
4-48 h. After incubation, the lowest concentration of sation reaction using aniline as catalyst (Xia et al.,
the compounds that completely inhibits macroscopic 2008). After protection of the phenolic hydroxy group
growth was determined and reported as MICs. by acetylation (Ac-FA, ), esterification was carried
The minimum biofilm eradication concentration out by reaction of corresponding acyl chlorides with
was inoculated to a two-fold diluted solution of the
3
1)
o
2
2
4
TM
(
MBEC) Assay for high-throughput screening (HTP; appropriate phenols to afford esters in moderate to
Innovotech) was obtained from the manufacturer and high yields (5a-e). The removal of the acetyl group
the Calgary Biofilm Device Method (Ceri et al., 1999) without the cleavage of the ester bond was performed
was performed to determine the MBEC values for using hydrazine monohydrate at room temperature

1256
B. Ç. Ergün et al.
(Nomura et al., 2002) to obtain target ester derivati-
ves of FA (6a-e). Structure elucidation of the title
compounds were verified by spectroscopic techniques
1
including H-NMR, IR and HRMS (LC-MS-TOF). All
FA analogs were found to have trans (
E
) configuration
confirmed by H-NMR spectra in that the coupling
constants of -H and -H on double bonds were 15.6-
6.0 Hz. The purity of the compounds were tested by
1
α
β
1
UPLC with UV detection and determined as 97-100%
pure before biological testing. The synthetic route to
the desired compounds is depicted in Fig. 1.
Antioxidant activity
In the present study, the antioxidant and radical
scavenging effects of the synthesized compounds (5a-
e, 6a-e) were determined with different in vitro test
systems in order to compare the antioxidant potential
of compounds for each method. The evaluation of the
ester derivatives was carried out at various concen-
Fig. 1. Synthetic pathways of aromatic ester derivatives of
ferulic acid
trations with comparison to α-tocopherol, an important
antioxidant in living systems. The compounds 5a-e radical scavenging activity of FA and its aromatic
were OH-protected hydrophobic molecules while the ester derivatives with different phenols (6a-e) were
compounds 6a-e were molecules carrying free phenolic examined and compared (Table II). FA (
3) showed
groups.
appreciable radical scavenging activity at the concen-
tration range of 0.0625 mM to 1 mM. Further coupling
DPPH free radical scavenging assay
of FA with differently substituted phenols (6a-e) also
DPPH is a stable free radical and accepts an elec- resulted in inhibition of DPPH activity although they
tron or a hydrogen radical to become a stable diama- were not as active as FA at the concentration ranges
gnetic molecule and is the most used standard assay tested. However, compounds that were OH-protected
in antioxidant activity studies which provides rapid
(5a-e) showed weak suppressive activities even at the
screening for radical scavenging activities of various highest concentrations (1 mM). As seen in Table II,
compounds (Vijay Kumar and Naik, 2010). DPPH free only the aromatic ester derivatives with free phenoxy
Table II. IC50 values of various ferulic acid derivatives for superoxide anion radical and DPPH radical scavenging
a
capacities and inhibitory effects on lipid peroxidation
Superoxide radical
scavenging capacity
DPPH radical
scavenging capacity
%
Inhibition of lipid peroxidation
Compounds
FA
(IC50, mM)
(IC50, mM)
1 mM
0.1 mM
3
(
)
NA
NA
0.10 ± 0.04
NA
NA
0.15 ± 0.05
NA
0.13 ± 0.07
NA
0.13 ± 0.02
NA
0.14 ± 0.04
NA
0.14 ± 0.03
0.013 ± 0.005
51 ± 20
11 ± 40
49 ± 20
34 ± 50
40 ± 20
25 ± 50
35 ± 10
3 ± 2
10
09
12
4
5a
0.61± 0.01
0.43 ± 0.02
0.46 ± 0.05
0.56 ± 0.02
0.57 ± 0.03
0.55 ± 0.02
0.19 ± 0.04
0.27 ± 0.01
0.75 ± 0.03
0.20 ± 0.02
0.51 ± 0.05
6a
25
5
6
b
b
NA
NA
NA
NA
NA
NA
NA
NA
52 ± 3
5
6
c
c
5
6
d
d
25 ± 60
22 ± 30
5 ± 1
5
6
e
e
2 ± 3
α
-tocopherol
73 ± 20
a
NA: Not Active; Each value represents the means ± S.D. of three independent experiments for determination of IC50 values.

Biological Activity of Ferulic Acid Derivatives
1257
groups (6a-e) and FA itself showed DPPH radical several Gram-positive, Gram-negative bacterial strains
scavenging abilities (IC50 values of 0.1-0.15 mM), indi- including drug-resistant isolates and several Candida
cating that the phenolic moiety present in the mole- strains. The MIC values were determined by a micro-
cular structure was responsible for the DPPH radical dilution method in MHB and RPMI-1640 medium for
scavenging activity. The presence of hydrophobic the antibacterial and antifungal assays, respectively.
branched alkyl groups (6a-b), electron withdrawing According to the data (Table III), most of the com-
(6c) and electron donating (6d-e) substituents on the pounds showed better antibacterial activity against
aromatic ester moiety did not significantly influence Gram-positive bacteria S. aureus E. faecalis and their
,
the DPPH radical scavenging activity compared to FA. drug-resistant isolates than other tested Gram-nega-
tive bacteria. The results revealed that compounds 6b
Superoxide anion radical scavenging assay
and 6c displayed significant activity against E.
The inhibitory effects of FA, Ac-FA and their ester faecalis with MIC values of 16 µg/mL for each com-
derivatives against O 2• − at different concentrations pound. Additionally, these two derivatives had equal
were tested and calculated IC50 values are given in to or better activity than gentamycin and vancomycin
Table II. As shown, FA (
any measurable activity to suppress superoxide gen- 32 and for 6c, 16
eration measured by cytochrome c reduction (inhibi- strated significant activity against S. aureus with a
tion of superoxide generation was 25% for FA and MIC value of <8 g/mL. All other compounds were
3% for Ac-FA at 1 mM). However, all tested ester moderately active for all tested strains including drug-
derivatives regardless of the free phenolic group (5a-e resistant types. Notably, all of them had better activi-
3
) and Ac-FA (
4
) did not show against vancomycin-resistant E. faecalis (MIC for 6b
,
µg/mL). Compound 6c also demon-
µ
4
•−
2
and 6a-e) showed significant O -scavenging capaci- ties than gentamycin against drug-resistant E. coli
ties at concentration ranges between 0.05 and 1 mM and had comparable activities to gentamycin against
and the IC values were determined from the calibra- drug-resistant K. Pneumoniae. On the other hand,
50
tion curve for each compound (Table II). In particular, compounds 5a-c and 6c had noticeable antifungal
5d
(IC50 = 0.19 mM), 6d (IC50 = 0.27 mM) and 6e (IC50 activities against Candida strains (Table III). Com-
=
0.2 mM) had approximately two-fold higher activi- pounds 6b and 6c had comparable activities with
ties as compared to
other derivatives also showed comparable IC50 values each compound and fluconazole). Compounds 5a and
between 0.43 and 0.75 mM) to -tocopherol. 5c were moderately active against C. parapsilosis and
C. albicans
α-tocopherol (IC50 = 0.51 mM). All fluconazole for C. krusei (MIC values of 64 µg/mL for
(
α
.
LP assay
In the LP process, the initiation of a peroxidation Bacterial biofilm eradication activity
sequence refers to attack of a reactive oxygen species
In human medicine, drug-resistant nosocomial in-
•
(ROS), such as hydroxyl radical (OH ), which easily fections caused by staphylococci have been associated
generates free radicals from polyunsaturated fatty with the growth of these bacteria in biofilms which
•
acids. OH is known as the most efficient ROS to are known as the most predominant form with high
initiate LP (Caliskan-Ergün et al., 2008). In the pre- resistance to traditional antibiotics (Dunne, 2002;
sent study, the LP system forms MDA which can be Vuong and Otto, 2002; Melchior et al., 2006). Once
•
readily detected by TBA and was used to assess OH
formed, biofilms are difficult to remove as they are 10-
formation. Using the LP assay, FA, Ac-FA and their 1000 times more resistant to antimicrobial agents,
ester derivatives showed rather weak inhibitory effects when compared with planktonic growing (free float-
on MDA formation at 1 mM and 0.1 mM. Except for ing) bacteria of the same strain (Amorena et al., 1999;
compounds
was observed concerning inhibition of LP (51%, 49%, ester derivatives (5a-d
0% and 35% at 1 mM, respectively; Table II). These inhibit biofilm formation by S. aureus and its isolate,
3
(FA) and 5a-c, no significant activity Olson et al., 2002). For these reasons, we also tested 8
,
6a-d) for their ability to
4
results may suggest that the tested FA derivatives in E. faecalis and its isolate, C. krusei and C. parapsilo-
general might be important as potent superoxide sis. The results are reported as the MBEC along with
radical scavengers rather than hydroxyl radical scav- MIC values for planktonic bacteria in Table III for
engers under in vitro conditions.
comparison. Interestingly, compound 6c caused an
inhibition of biofilm formation by S. aureus with an
Antimicrobial activity
MBEC value of <8 µg/mL which was 64 and 128 times
All the newly synthesized FA derivatives were more potent than ofloxacin and vancomycin, respecti-
assayed in vitro for antimicrobial activity against vely. We also demonstrated that compounds 5b 5c
,
,

1258
B. Ç. Ergün et al.
Table III. The antimicrobial, antifungal and antibiofilm activities of the synthesized compounds and control drugs
Microorganisms
Compound
Gram-negative
Gram-positive
Fungi
Ck
Methoda
Ec
Ecb
Kp
Kpb
Sa
Sab
Ef
Efb
Ca
Cp
3
(
4
5
FA
)
MIC
MIC
MIC
MBEC
MIC
MBEC
MIC
MBEC
MIC
MBEC
MIC
MBEC
MIC
MBEC
MIC
MBEC
MIC
MBEC
MIC
MIC
MIC
MIC
MIC
MBEC
MIC <0.0625
256
256
128
-
256
-
256
-
256
-
256
-
256
-
256
-
256
-
256
256
256
-
256
-
256
-
256
-
256
-
256
-
256
-
256
-
256
256
>2048
1024
-
-
64
-
256
256
128
-
256
-
256
-
128
-
256
-
128
-
256
-
256
-
256
256
2
0.5
-
-
256
256
256
-
256
-
256
-
256
-
256
-
256
-
256
-
256
256
128
512
256
512
128
512
128
512
128
512
<8
256
256
256
>1024
256
>1024
128
>1024
128
>1024
256
>1024
128
>1024
256
>1024
256
>1024
256
256
>2048
32
128
128
128
1024
128
1024
128
1024
16
1024
128
1024
16
1024
128
1024
256
1024
128
128
1
128
128
128
1024
128
1024
128
1024
32
1024
128
1024
16
1024
128
1024
128
1024
128
128
0.5
128
128
128
-
128
-
128
-
64
-
64
-
64
-
128
-
128
-
128
128
-
-
-
-
-
-
1
0.25
-
128
128
128
1024
128
1024
128
64
128
128
64
a
>1024
128
>1024
128
>1024
128
>1024
128
>1024
64
>1024
128
>1024
128
>1024
128
128
-
-
-
-
-
-
1
1
32
6
a
5
b
b
6
64
256
128
64
64
64
128
64
128
64
128
128
-
-
-
-
-
-
64
2
32
5
c
c
6
<8
5
d
d
256
512
256
512
256
256
0.5
6
256
-
256
256
>2048
256
5
6
e
e
256
256
2
1
-
Ampisilin
Gentamisin
Vankomisin
0.125
1
>1024 >1024
0.125
512
4
1
1024
1
32
32
1024
4
1024
-
-
-
1
-
Ofloksasin
Flukonazol
AmfoterisinB MIC
MBEC
0.125
0.5
0.25
>1024 >1024
MBEC
MIC
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
Abbreviations: Ec
,
Escherichia coli ATCC 25922; Kp
,
Klebsiella pneumoniae RSKK 574; Sa
,
Staphylococcus aureus ATCC
29213; Ef
,
Enterococcus faecalis ATCC 29212; Ca
,
Candida albicans ATCC 10231; Ck
,
C. krusei ATCC 6258; Cp C.
,
parapsilosis ATCC 90028.
a
MIC, Minimal inhibitory concentration (
µ
g/mL); MBEC, minimal biofilm eradication concentraion (
K. pneumoniae isolate ESBL (resistant to beta lactam
, S. aureus isolate MRSA (resistant to beta lactam antibiotics); E. faecalis izolat (VRE) (resistant to
µg/mL)
b
Ec, E. coli isolate ESBL (resistant to beta lactam antibiotics); Kp,
antibiotics); Sa
vancomycin).
5
d
,
6c and 6d had comparable MBEC values (64
µg/ ity in the DPPH assay with respect to IC50 values of
mL) to amphotericin B (32 g/mL) to inhibit the bio- the ester derivatives with a free phenolic group seem-
film formation by C. krusei (Table III).
µ
ed to have similar activity. In a homogenous solution,
modification of the carboxylic acid side chain of FA by
esterification with different phenols does not contri-
bute to the antioxidant potency of FA through in-
DISCUSSION
Because the main mechanism of action of the phe- tramolecular interactions between the side chain and
nolic antioxidant, FA, is considered to be the scaveng- the phenoxy nucleus. Therefore, the important struc-
ing of free radicals (Graf, 1992), the reactivity of FA tural feature for DPPH radical scavenging is the pre-
derivatives (5a-e, 6a-e) was initially tested toward the sence of the free phenolic group and not the nature of
stable radical DPPH. Evaluation of antioxidant activ- the aromatic ester chain in the tested compounds.

Biological Activity of Ferulic Acid Derivatives
1259
•
−
O
has been implicated in several pathophysiolo- studies have shown that biofilm-grown microorganisms
2
gical processes due to its transformation into more have an inherent lack of susceptibility to antimicro-
•
•−
reactive species including OH which initiates LP. O
2
bial agents as compared to planktonic cultures of the
has also been known to directly initiate LP which sig- same organism (Costerton et al., 1995, 1999; Wilson,
nificantly contributes to the development of pathologi- 1996; Ceri et al., 1999; Ali et al., 2006). However, this
cal disorders such as atherosclerosis (Halliwell, 1994). resistance is lost once conditions are reverted to allow
In the present study, we demonstrated that all com- planktonic growth (Costerton et al., 1995). In the
pounds regardless of free phenolic nucleus caused present study, the biofilm formed by S. aureus strain
•−
significant attenuation of O
2
generation. Therefore, proved to be significantly more resistant to antibiotics
we can clearly say that the presence of phenolic group than planktonic cultures of the same microorganism
as well as the nature of the aromatic ester chain (MBECs were 1,000-fold or greater than the MICs for
affects activity since some of the derivatives resulted vancomycin and ofloxacin). However, the MBEC value
•−
in potent inhibition of O2 (Table II). In particular, the of 6c fell to within the same range of susceptibility for
presence of a 4-methoxy-phenyl on the aromatic ester MIC assay indicating that this compound had a
•−
moiety strikingly affected the O
2
scavenging activity marked effect for eradication of biofilms formed by S.
as compared to -tocopherol. Taken together, all com- aureus as compared to reference antibiotics. This dif-
α
•−
pounds exhibited a scavenging effect on O2 genera- ference in antimicrobial susceptibility between plank-
tion that could help to prevent or ameliorate oxidative tonic and biofilm populations may be explained either
damage. However, with regard to inhibition of LP, by differences in the diffusion of 6c and control anti-
none of the compounds demonstrated appreciable biotics (Stewart, 1996) or as a result of more complex
activity as demonstrated in rat liver homogenates changes in the microbial physiology of the biofilm
used as a membrane model (Caliskan-Ergün et al., (Costerton et al., 1995; Stewart, 1996; Davies et al.,
2
008). Since these differences in antioxidant potency 1998); this needs to be investigated further.
between homogenous and heterogeneous phases were In conclusion, we successfully demonstrated that
difficult to reconcile, we realized that the latter may simple aromatic esters of FA (5d 6d-e) showed signi-
,
•−
be explained by an insufficient anchorage of the side ficant O2 scavenging properties providing useful
chain with membrane phospholipids. It is very likely information for the design of new chemical antioxid-
that the anchorage of the aromatic ester side chain ant entities with protective activities. Furthermore,
would negatively affect the positioning of the radical antimicrobial evaluation of some of the presented
scavenging nucleus of the phenoxy moiety constrain- structures (6b and 6c) against Gram-positive bacteria
ing the phenolic nucleus outside of the bilayer surface
to quench the flux of the free oxyradicals generated by and fungi (Candida strains) are encouraging since
(S. aureus, E. faecalis and their drug-resistant strains)
+3
the Fe -ascorbic acid system. This is supported by a compound 6c also demonstrated potent MBEC values
previous study where anchoring of molecules within which was comparable to its MIC value for planktonic
the phospholipids membrane is favored when the side cultures, and was 64 and 128 times more potent than
chain on alkyl esters of FA has a high degree of ofloxacin and vancomycin, respectively. Further studies
conformational flexibility which enables to bend in a with these types of compounds are under ongoing
loop conformation toward the ethylenic part of FA investigation in our laboratory.
(Anselmi et al., 2004). Since our molecules are expect-
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