Synthesis and biological activities of new Mannich bases of chlorokojic acid derivatives

Article abstract of DOI:10.1007/s00044-010-9338-x

A novel series of 6-chloromethyl-3-hydroxy-2- substituted 4H-pyran-4-one derivatives were synthesized and tested for their antibacterial, antifungal and antiviral in vitro properties. In the view of activity results, compounds 8-11 (MIC: 8 μg/ml) were more remarkably active against Staphylococcus aureus and Enterococcus faecalis. Compounds 1-7 were highly active against Candida albicans and C. parapsilosis with MIC value of 8 μg/ml. Compound 9 bearing 3-chlorophenyl moiety was determined to be the most active compound against RNA virus PI-3.

Full text of DOI:10.1007/s00044-010-9338-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:443–452
DOI 10.1007/s00044-010-9338-x
ORIGINAL RESEARCH
Synthesis and biological activities of new Mannich bases
of chlorokojic acid derivatives
¨
Mutlu Dilsiz Aytemir Berrin Ozc¸elik
•
Received: 11 May 2009 / Accepted: 3 March 2010 / Published online: 18 March 2010
Ó Springer Science+Business Media, LLC 2011
Abstract A novel series of 6-chloromethyl-3-hydroxy-2-
substituted 4H-pyran-4-one derivatives were synthesized
and tested for their antibacterial, antifungal and antiviral in
vitro properties. In the view of activity results, compounds
8–11 (MIC: 8 lg/ml) were more remarkably active against
Staphylococcus aureus and Enterococcus faecalis. Com-
pounds 1–7 were highly active against Candida albicans
and C. parapsilosis with MIC value of 8 lg/ml. Compound
9 bearing 3-chlorophenyl moiety was determined to be the
most active compound against RNA virus PI-3.
frequent pathogen, are the most widespread and threatening
fungal pathogens today, and responsible for the majority of
invasive and non-invasive fungal infections. The emer-
gence of organisms resistant to nearly all the class of
antimicrobial agents has become a serious public health
concern. To overcome the drawbacks of the current anti-
fungal drugs and obtain more efficacious drugs, an anti-
fungal drug having a novel mode of action should be
developed (Thomas and Patterson, 2002). On the other
hand, Herpes simplex virus Type-1 (HSV-1) is known as a
very common human pathogen that causes a variety of
illnesses. Influenza virus infection is also common and it
may cause life-threatening events in high-risk patients.
Eventually, there is a need for safe and effective antiviral
agents in the management of the signs and symptoms of
Herpes simplex and influenza virus infections (Esquenazi
Keywords Chlorokojic acid ꢀ Kojic acid ꢀ
Mannich bases ꢀ Antimicrobial ꢀ Antiviral
Introduction
¨
et al., 2002; Ozc¸elik et al., 2009).
The development of antimicrobial agents to treat infections
has been one of the most notable medical achievements of
the past century. However, these advances in medical care
are threatened by a natural phenomenon known as ‘‘anti-
microbial resistance’’. Antimicrobial resistance, whereby
microbes mutate and become less susceptible to these
‘‘miracle drugs’’ over time, creates problems to physicians
for infectious diseases, as they must work quickly to
determine what drug will work on a patient’s infection.
Candida species, especially C. albicans which is the most
Some natural antibiotics contain a siderophore structure,
e.g., albomycin. Siderophores are low-molecular weight
compounds manufactured by microorganisms to facilitate
the uptake of iron(III). They contain either hydroxamate or
catechol groups, which are able to sequester iron cations
(Hider et al., 1992). Chelating ability of kojic acid
(5-hydroxy-2-hydroxymethyl-4H-pyran-4-one), which has
a catechol-like function, has been used for analytical pur-
poses, also plays a significant role in its antibacterial and
antifungal activities (Weinberg, 1957; Synytsya et al.,
2008; Fassihi et al., 2009). Also, metal-binding compounds
which were formed by the reaction of hydroxypyranone
derivatives with metallic ions have proven activities in
fungal infectious (Brtko et al., 2004).
M. D. Aytemir (&)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Hacettepe University, 06100 Sihhiye-Ankara, Turkey
e-mail: mutlud@hacettepe.edu.tr
Previous antimicrobial activity studies showed that kojic
acid and its derivatives have antibacterial activity (Kotani etal.,
1975; Pirselova et al., 1996; Aytemir et al., 2003; Bentley,
2006; Fassihi et al., 2009). Moreover, 2-chloromethyl-5-
¨
B. Ozc¸elik
Department of Pharmaceutical Microbiology, Faculty
of Pharmacy, Gazi University, 06330 Ankara, Turkey
123

444
Med Chem Res (2011) 20:443–452
hydroxy-4H-pyran-4-one (chlorokojic acid) inhibited Aer-
omonas aerogenes, Micrococcus pyogenes var. aureus,
Samonellal typhosa, Penicilium digitalum, Russula nigricans
and Saccharomyces cerevisiae (Wolf and Westveer, 1950).
Brtko and co-workers reported that chlorokojic acid and other
halogen derivatives have significant antifungal activity. Also,
their copper(II) salts’ complex derivatives were prepared and
found to be more active than chlorokojic acid (Brtko et al.,
2004). It was shown that kojic acid derivatives were deter-
mined as having significant antifungal effects against different
Candida species and Pythium graminicola by the different
researchers (Kayahara et al., 1990; Aytemir et al., 2003, 2004;
Fassihi et al., 2009). According to its antibacterial and fungi-
cidal properties, kojic acid is used as a food additive to prevent
browning (Burdock et al., 2001; Brtko et al., 2004; Bentley,
2006). Moreover, kojic acid derivatives exhibit a number of
interesting bioactivities including herbicidal (Veverka and
Kralovicova, 1990), anti-speck (Uchino et al., 1988), pesticide
and insecticide (Alverson, 2003), antitumor activity (Higa
et al., 2007), anti-diabetic (Xiong and Pirrung, 2008), antiep-
ileptic (Aytemir et al., 2004, 2010), modest anti-inflammatory
effect (Brtko et al., 2004), and as a skin-whitening product in
cosmetic (Burdock et al., 2001; Bentley, 2006).
Literature survey reveals that piperazine ring has an
important factor for antimicrobial activity. Eperezolid,
posaconazole, ketoconazole and itraconazole, which are
currently important antimicrobial drugs used for the
treatment of microbial infections, contain a piperazine
ring in their structures (Upadhayaya et al., 2004; Forou-
madi et al., 2006). In the framework of this study, we aim
to synthesize new Mannich bases of chlorokojic acid
derivatives having substituted piperazine ring in order to
develop new antimicrobial and antiviral compounds
(Table 1).
Table 1 Yields, melting points and elemental analysis results of the synthesized compounds 1–11
O
OH
CH2
R
ClCH2
O
N
N
Comp. no.
R
M.p. (°C)
Yield (%)
Empirical formula
MW
Elemental Anal. Calc./Found (%)
C
H
N
1
3-CF3
4-CF3
2-F
153-4
167-8
148-9
154-5
125-6
136-7
165-6
137-8
151-2
174-5
160-1
82
88
71
65
54
79
92
50
74
86
83
C18H18ClF3N2O3
C18H18ClF3N2O3
402.10
402.10
352.09
352.09
364.11
364.11
364.11
368.06
368.06
368.06
402.03
53.67
53.90
53.67
53.51
57.88
57.85
57.88
57.53
59.26
59.54
59.26
59.04
59.26
59.45
55.30
54.73
55.30
54.95
55.30
54.55
50.58
50.12
4.50
4.01
4.50
4.47
5.14
5.11
5.14
5.10
5.80
5.83
5.80
5.72
5.80
5.77
4.91
4.80
4.91
4.85
4.91
4.61
4.24
4.27
6.95
6.88
6.95
6.92
7.94
7.84
7.94
7.80
7.68
7.62
7.68
7.57
7.68
7.65
7.59
7.41
7.59
7.47
7.59
7.58
6.94
6.80
2
3
C
C
17H18ClFN2O3
17H18ClFN2O3
4
4-F
5
2-OCH3
3-OCH3
4-OCH3
2-Cl
C18H21ClN2O4
C18H21ClN2O4
C18H21ClN2O4
6
7
8
C
C
C
C
17H18Cl2N2O3
17H18Cl2N2O3
17H18Cl2N2O3
17H18Cl3N2O3
9
3-Cl
10
11
4-Cl
3,4-di-Cl
123

Med Chem Res (2011) 20:443–452
Results and discussion
Chemistry
445
chlorokojic acid. Additionally, 6-morpholino or piperidi-
nomethyl chlorokojic acid were prepared via Mannich
reaction (O’brien et al., 1960). At latter study, Ichimoto
and Tatsumi (1965) synthesized Mannich bases of kojic
acid and pyromeconic acid in either acidic or basic med-
ium, giving details of mechanism, using aliphatic or het-
erocyclic secondary amines such as dimethylamine,
diethylamine or morpholine, respectively.
Kojic acid provides a promising skeleton for development
of new more potent derivatives such as chlorokojic acid,
allomaltol and pyromeconic acid (Fig. 1). They are good
ligands for the nucleophilic and electrophilic substitution
reactions (Ichimoto and Tatsumi, 1965; Uher et al., 2000).
Therefore, many researchers used them for starting or
intermediate material for preparation of new compounds
(O’brien et al., 1960; Ichimoto and Tatsumi, 1965; Ellis
et al., 1996; Aytemir et al., 2003, 2004; Dehkordi et al.,
2008).
The 6-chloromethyl-3-hydroxy-2-(substituted phenyl-
piperazin-1-yl)methyl-4H-pyran-4-one derivatives (1–11)
were prepared by the reaction of appropriate substituted
phenyl piperazine derivatives with chlorokojic acid and
formaline at room temperature as shown in Scheme 1. The
derivatives of chlorokojic acid were obtained in good
yields. The structures of the synthesized compounds 1–11
In this study, chlorokojic acid was used as the starting
material. The enolic structure of neutral chlorokojic acid is
expected to be the most stable one (Zborowski et al.,
2004). It was synthesized from commercially available
kojic acid in one-step reaction according to literature.
Chlorination of 2-hydroxymethyl moiety of kojic acid
using clean thionyl chloride at room temperature afforded
chlorokojic acid (Ellis et al., 1996; Aytemir et al., 2003,
2004).
1
were identified by using IR, H NMR, ESI-MS data and
elemental analysis. Yields, the melting points and ele-
mental analysis of the synthesized compounds are pre-
sented in Table 1. IR spectra of 1–11 showed stretching
bands associated with C=O, C=C and C–O stretching at
1642–1622, 1597–1429 and 1218–1162 cm-1, respec-
1
tively. When the H NMR spectra was investigated for the
all synthesized compounds, characteristic singlet peaks of
the 4H-pyran-4-one (H5) ring proton were found in the
region 6.45–6.63 ppm in accordance with literatures (Ellis
et al., 1996; Aytemir et al., 2003, 2004). Substituted
piperazine ring protons were found at 2.50–2.71 and
2.61–3.29 ppm as two triplet peaks. Also, signals of
ClCH2– group protons were displayed as singlet peaks at
4.55–4.73 ppm. The methylene group protons of 1–11
appeared as singlet peaks at 3.51–3.72 ppm. The ESI mass
spectra of all compounds showed suitable peaks in accor-
dance with previously study (Aytemir et al., 2010).
Because of phenol-like property of kojic acid, it readily
undergoes aminomethylation in the Mannich reaction ortho
to enolic hydroxyl group. It was reported that di-Mannich
derivatives which were formed at 3- and 6-positions,
obtained in an acidic medium by the reaction of kojic acid,
formaline and aromatic amine derivatives. However,
O’brien et al. (1960) showed that derivatives of Mannich
bases occured at only 6-position of kojic acid, which were
synthesized using dimethylamine, diethylamine, pyrroli-
dine, morpholine, piperidine or 4-methylpiperazine and
Fig. 1 Hydroxypyranone
derivatives
O
O
O
O
OH
OH
OH
OH
HOCH2
CH3
ClCH2
O
O
O
O
Pyromeconic acid
Allomaltol
Chlorokojic acid
Kojic acid
Scheme 1 Schematic
representation of the
synthesized compounds. R =
3-Trifluoromethyl-,
4-trifluoromethyl-, 2-fluoro-,
4-fluoro-, 2-methoxy-,
3-methoxy-, 4-methoxy-,
2-chloro-, 3-chloro-, 4-chloro-,
3,4-dichloro-, (a) SOCl2,
(b) MeOH, r.t. room
O
O
OH
OH
R
a
HCHO
HN
N
HOCH2
ClCH2
Chlorokojic acid
O
O
r.t.
Kojic acid
b
O
temperature
OH
R
ClCH2
N
N
CH2
O
123

446
Med Chem Res (2011) 20:443–452
In vitro antibacterial and antifungal activity
compounds have similar activity against to E. coli. Fur-
thermore, compounds 1, 2, 7 and 11 (MIC: 16 lg/ml)
showed high activity against P. aeruginosa in the entire
series. Investigation among other Gram-negative bacteria,
compounds 1, 3, 4, and 7 and compounds 2 and 7
were more active derivatives against K. pneumoniae
and A. baumannii, respectively, than the others (MIC:
16 lg/ml). Also, compounds 1, 2 and 4 (MIC: 16 lg/ml)
were the most effective compounds towards P. mirabilis.
According to the obtained data in Table 3, it can be
considered that structural differences on the substitution at
2-position of hydroxypyranone nucleus changed their
activity against S. aureus but not its isolated strains of
methicillin-resistant S. aureus (MRSA). The antibacterial
activity of the synthesized compounds 8–11 possessing a
chloro-substituent on phenyl ring were found to have sig-
nificantly high activity against Gram-positive bacteria such
as S. aureus and E. faecalis showing a bacterial inhibition
8 lg/ml. On the other hand, the other synthesized com-
pounds caused slight activity against isolated strains of
S. aureus. As structure–activity relationship (SAR), while,
methoxy substitution in the ortho, meta and para position
of the phenyl rings (5–7) have less activity but the chloro,
fluoro and trifluoromethyl substitution (2–4 and 8–11) of
The antibacterial and antifungal activity profiles of the
newly synthesized compounds were assessed for antimi-
crobial activity against both standards and the isolated
strains of microorganism. Tables 2 and 3 describe the in
vitro antimicrobial activity with the MIC values of com-
pounds 1–11. According to our data (Tables 2 and 3), the
synthesized compounds showed a broad spectrum of
activity with MIC values 8–32 lg/ml against Gram-posi-
tive and Gram-negative standard strains. In the meantime,
the synthesized compounds showed moderate effect with
MIC values 32 to C128 lg/ml against drug-resistant iso-
lated both of strains.
As given in the Table 2, antibacterial activity of com-
pounds 1–4, showed better activity against standards
strains of Gram-negative bacteria like E. coli, P. aerugin-
osa, P. mirabilis and K. pneumoniae. As for Gram-positive
bacteria, MIC values 8–64 lg/ml and 64 to C128 lg/ml
were determined for standard and isolated drug-resistant
strains, respectively. Compounds 1–4 which bearing fluoro
and trifluoromethyl-substituents on phenyl rings have more
potent activity (MIC: 16 lg/ml) among this Mannich base
derivatives series towards E. coli. The other synthesized
Table 2 Antibacterial activity of the synthesized compounds 1–11 and the control drugs (MIC in lg/ml)
Comp. no.
Gram-negative standard and clinic isolated strains
E. coli
P. aeruginosa
P. mirabilis
K. pneumoniae
A. baumannii
ATCC
35218
Isolated strain
ESbL?
ATCC
10145
Isolated
strain
ATCC
7002
Isolated strain
ESbL?
RSKK
574
Isolated strain
ESbL?
RSKK
02026
Isolated
strain
1
16
16
16
16
32
32
32
64
32
64
64
2
64
64
16
16
32
32
32
32
16
64
32
64
16
–
64
64
16
16
64
64
16
32
64
64
32
16
32
32
32
32
16
64
32
64
64
2
64
64
2
3
64
64
32
64
16
64
64
4
64
64
16
64
16
64
64
5
64
64
32
64
32
64
64
6
64
64
32
64
32
64
64
7
64
64
32
64
16
32
32
8
[128
64
[128
64
64
[128
64
64
[128
64
[128
64
9
32
32
10
11
AMP
VAN
GM
OFX
LVX
[128
64
[128
64
64
[128
64
64
[128
64
[128
32
32
32
[128
–
–
2
[128
–
2
[128
–
[128
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0.12
\0.12
0.5
0.5
1
64
\0.12
\0.12
1
\0.12
\0.12
0.5
1
0.12
0.12
64
1
64
1
64
AMP ampicilline, VAN vancomisine, GM gentamicin, OFX ofloxacin, LVX levofloxacine
– No activity observed, E. coli isolates; (resist to trimethoprim–sulfamethoxazole, cefepime, tazobactam), P. aeruginosa isolates
(resist to trimethoprim–sulfamethoxazole, tazobactam), P.mirabilis isolates (resist to trimethoprim–sulfamethoxazole, cefepime, tazobactam),
K. pneumoniae isolates (resist to trimethoprim–sulfamethoxazole, amoxicillin clavulonat, cefriaxon, cephepim, aztreonam) A. baumannii isolates
(resist to trimethoprim–sulfamethoxazole, cefepime)
123

Med Chem Res (2011) 20:443–452
447
Table 3 Antibacterial and antifungal activities of the synthesized compounds 1–11 and the control drugs (MIC in lg/ml)
Comp. no.
Gram-positive standard and clinic isolated strain bacteria
Bacteria
Fungi
S. aureus
E. faecalis
B. subtilis
C. albicans
C. parapsilosis
ATCC
25923
Isolated
strain MRSA
ATCC
29212
Isolated
strain
ATCC
6633
Isolated
strain
ATCC
10231
ATCC
22019
1
32
16
16
16
64
64
32
8
64
64
16
8
32
32
32
8
64
64
16
32
32
32
64
[128
32
[128
64
0.5
–
8
8
8
8
2
3
64
16
16
64
64
32
8
32
8
8
8
4
64
32
16
16
16
32
16
16
16
16
0.12
–
8
8
5
128
128
64
128
128
64
8
8
6
8
8
7
8
8
8
[128
128
[128
64
[128
128
[128
64
16
16
16
16
–
16
16
16
16
–
9
8
8
10
8
8
11
8
8
AMP
VAN
GM
OFX
LVX
KET
FLU
\0.12
0.12
–
[128
2
0.5
–
[128
–
–
–
–
–
–
–
–
–
–
0.25
0.25
–
64
1
32
–
–
–
–
128
–
0.5
–
32
–
–
–
–
–
–
–
1
1
–
–
–
–
–
–
2
4
AMP ampicilline, VAN vancomisine, GM gentamicin, OFX ofloxacin, LVX levofloxacine, KET ketoconazole, FLU fluconazole
– No activity observed, S. aureus isolates (resist to oxacillin, gentamicin, aztreonam, trimethoprim–sulfamethoxazole), E. faecalis isolates (resist
to cephalosporins and beta-lactam), B. subtilis isolates (resist to ceftriaxon)
phenyl leads to increase in antibacterial activity towards
S. aureus (MIC: 8–16 lg/ml). In addition, there is no dif-
ference in antimicrobial activity with the location of the
chloro-substituent on phenyl ring. Their antibacterial
activity results were encouraging, although some com-
pounds (1, 5, 6 and 7) manifested moderate antibacterial
activity against standard strains of S. aureus. The anti-
bacterial potential against E. faecalis was exhibited by
compounds 2 and 8–11 at concentration 8 lg/ml among the
synthesized compounds. Beside, compounds 8–11 were
found to be active against Gram-positive while they were
less effective towards Gram-negative bacteria. The other
compounds had manifested moderate inhibitory activity
against E. faecalis. Moreover, compounds 2 and 3, which
bears trifluoromethyl- and fluorophenylpiperazinemethyl
moiety at the 2-position of pyran-4-one ring, were the most
active derivatives against standard strains of B. subtilis
with a MIC value of 8 lg/ml.
methoxy phenyl derivatives, were determined to posses
high antifungal potential against C. albicans and C. par-
apsilosis when compared to other tested compounds. These
compounds had comparable results with fluconazole (MIC:
4 lg/ml) against C. parapsilosis.
Antiviral activity
In this study, our goal was to investigate the role of effi-
cient substitution on different position of phenylpiperazine
ring. All compounds were assayed against both HSV-1 and
PI-3 by using Madin–Darby Bovine Kidney and Vero cell
lines with the aim of to capture structure relationship in
each of the compounds. The results of the antiviral study
are presented in Table 4.
As given in CPE inhibitory concentration ranging,
compound 7 bearing 4-methoxy substituent showed sig-
nificant activity against HSV-1 as potent as the reference
compound acyclovir, but limited activity at maximum and
minimum concentration ranges of 1.6 to \0.1 lg/ml with
the maximum non-toxic concentration (MNTC) value of
1.6 lg/ml. Additionally, compound 9 (0.8–0.1 lg/ml) was
As seen in the Table 3, all Mannich base derivatives
showed similar activity profile in fungi (MIC: 8–16 lg/ml).
The screening data indicates that especially compounds 1–
7 (MIC: 8 lg/ml), which have trifluoromethyl, fluoro and
123

448
Med Chem Res (2011) 20:443–452
Table 4 Cytotoxicity on MDBK and Vero cells as well as antiviral activity against HSV-1 and PI-3 results of the compounds 1–11
Comp. no.
MDBK cells
Vero cells
MNTCs (lg/ml)
CPE inhibitory concentration
MNTCs (lg/ml)
CPE inhibitory concentration
HSV-1
PI-3
Max.
Min.
0.05
Max.
Min.
1
0.8
0.8
0.8
0.8
0.8
0.8
1.6
1.6
1.6
1.6
1.6
1.6
–
0.8
0.8
0.8
0.8
0.8
0.8
1.6
0.4
0.8
0.4
0.4
1.6
–
0.8
0.4
0.8
1.6
1.6
1.6
1.6
0.4
0.8
0.4
0.4
–
0.4
0.4
0.2
0.2
0.2
0.4
0.8
0.4
0.8
0.4
0.4
–
0.025
0.025
0.025
0.025
0.05
2
0.05
0.05
0.05
0.2
3
4
5
6
0.2
0.1
7
0.1
0.05
8
0.1
0.2
9
0.1
0.025
0.025
0.05
10
0.1
11
0.1
Asiklovir
Oseltamivir
\0.012
–
–
1.6
1.6
\0.012
MNTCs maximum non-toxic concentrations, CPE cytopathogenic effect, HSV-1 Herpes simplex virus Type-1, PI-3 Parainfluenza-3 virus,
Max. Maximum, Min. Minimum
– Not done, activity observed
shown anti-Herpes simplex activity but less potent. On the
other hand, compounds 1–4 were shown as same activity as
compound 7 but on higher non-toxic concentrations
(MNTC: 0.8 lg/ml). Among the tested Mannich bases
derivatives, compounds 5, 6, 8, 10 and 11 were less active
against DNA virus. Taken into account CPE inhibitory
concentration ranging against the RNA viruses PI-3,
compound 9 (0.8–0.025 lg/ml) had remarkable antiviral
activity in Mannich base derivatives. Thereto, compounds
7 and 1 were less active than compound 9. While the
activities of compounds 2 and 10 (0.4–0.025 lg/ml)
against PI-3 were in similar CPE inhibitory concentration
range, compounds 3, 4 and 11 had lower activity than those
had. Also, compounds 5 and 6 were negligible values as
seen in Table 4.
compound 9 was shown to display good antiviral activity.
While 4-chloro (10) and 3,4-dichloro (11) were less active,
2-chloro derivative was found to be inactive.
Investigation among MNTCs of synthesized compounds
1–11 on the Vero and MDBK cell line was found
0.4–1.6 lg/ml. As this result, compounds 1–6 (0.8 lg/ml)
were more toxic than compounds 7–11 (1.6 lg/ml) on
MDBK cell culture line. At this concentration, the cells did
not exhibit altered morphology or growth characteristics
indicative of cytotoxic effects. Thus, toxicity prevented the
evaluation of their potential antiviral effect at higher
concentration.
Conclusion
Concerning SAR, when the effects of mono or dichloro
substitution in the ortho, meta and para position of the
phenyl ring (8–11) were investigated, compound 9 contain-
ing 3-chloro-substituent was more active than 2-chloro,
4-chloro and 3,4-dichlorophenyl derivatives which showed
similar activity against HSV-1. Additionally, when the
effects of methoxy substituent were searched on activity
against both viruses, 4-methoxy derivative showed higher
activity than 2- and 3-methoxy derivatives. Also, changing
substitution of any position of trifluoromethyl (1, 2) and
fluoro (3, 4) phenyl derivatives were not effective on activity
towards both DNA and RNA viruses. Finally, evaluation of
effect on chloro-substitution against PI-3 infected Vero cell,
Further modification of the above structures may provide
compounds with better antibacterial and antiviral activities
among synthesized derivatives. In this study, the impact of
halogen substituent at phenylpiperazine moiety was
investigated. Different substituents on phenyl ring and
chelating effect were related with their antimicrobial
activity. The results of SAR studies indicated that the
presence of electron withdrawing groups is necessary for
the antimicrobial activity. Looking at the SAR between
hydroxypyranones, it can be concluded that the presence of
the halogen at the phenyl ring compounds 2 and 8–11
bearing chlorophenyl-moiety, have been found to enhance
123

Med Chem Res (2011) 20:443–452
449
6-(Chloromethyl)-3-hydroxy-2-({4-[3-
(trifluoromethyl)phenyl]piperazin-1-yl}methyl)-4H-pyran-
4-one (1)
the antibacterial activity towards S. aureus and E. faecalis.
Compounds 1 and 2 will have proved to be promising
antibacterial agents against Gram-negative bacteria in the
trifluoromethyl-moiety series. All of synthesized com-
pounds have better antibacterial effect against Gram-posi-
tive than Gram-negative bacteria. According to antifungal
potential, compounds 1–7 determined to be the most
remarkable active Mannich base derivatives against
C. albicans and C. parapsilosis. The synthesis studies
should be continued concerning this group of compounds
followed with further antimicrobial activity studies.
When the synthesized compounds were compared
within CPE inhibitory concentration ranges, compounds 1–
4 and 7 against HSV-1 and compound 9 against PI-3 were
appeared the most active derivatives as well as the controls.
They could be selected as lead compounds for the devel-
opment of novel antiviral agents.
IR (KBr disc) 1642 (C=O st), 1501 (C=C st) and
1
1162 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
2.71 (4H; t; piperazine), 3.29 (4H; t; piperazine), 3.72 (2H;
s; –CH2–), 4.73 (2H; s; 0ClCH2–), 6.63 (1H; s; H5), 7.11
0
(1H; d; J = 7.51; Ar0–H6 ), 7.21(1H; s; Ar–H2 ), 7.26 (1H;
0
d; J = 8.43; Ar–H4 ), 7.46 (1H; t; J = 7.98; Ar–H5 ),
9.41–9.48 ppm (1H; br; –OH). ESI m/e 195 (100%), 403
(M ? 1, 18.16%), 405 (M ? 3), 425 (M ? 23).
6-(Chloromethyl)-3-hydroxy-2-({4-[4-
(trifluoromethyl)phenyl]piperazin-1-yl}methyl)-
4H-pyran-4-one (2)
IR (KBr disc) 1635 (C=O st), 1523 (C=C st) and
1
1202 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
2.50 (4H; t; J = 4.00; piperazine), 2.61 (4H; t; J = 4.80;
piperazine), 3.63 (2H; s; –CH2–), 4.66 (2H; s; ClCH2–),
Experimental
0
0
6.55 (1H; s; H5), 7.04 (2H; d; J = 9.20; Ar–H2 , H6 ), 7.48
All chemicals used for the synthesis of the compounds were
supplied by Merck (Darmstadt, Germany) and Aldrich
Chemical Co. (Steinheim, Germany). Melting points were
determined by a Thomas Hoover Capillary Melting Point
Apparatus (Philadelphia, PA, USA) and were uncorrected.
IR spectra were recorded on a Jasco FT/IR-420 spectrom-
eter and Bruker Vector 22 IR (Opus Spectroscopic Software
Version 2.0) as KBr disc (c, cm-1). 1H NMR spectra were
obtained with a Varian 400 MHz spectrophotometer in
dimethylsulfoxide-d6 (DMSO-d6) and tetramethylsilane
(TMS) was used as an internal standard (chemical shift in d,
ppm). Mass analysis was carried out with a Micromass ZQ
LC–MS with Masslynx Software Version 4.1 by using ESI
(?) method. The elemental analyses were performed with a
Leco CHNS-932 (St. Joseph, MI, USA) at The Scientific &
Technological Research Council of Turkey-Ankara Testing
0
0
(2H; d; J = 8.40; Ar–H3 , H5 ), 9.20–9.30 ppm (1H; br;
–OH). ESI m/e 101 (100%), 403 (M ? 1, 23.65%), 405
(M ? 3), 425 (M ? 23).
6-(Chloromethyl)-2-{[4-(2-fluorophenyl)piperazin-1-
yl]methyl}-3-hydroxy-4H-pyran-4-one (3)
IR (KBr disc) 1623 (C=O st), 1499, 1453 (C=C st) and
1
1200 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
2.64 (4H; t; J = 4.40; piperazine), 3.01 (4H; t; J = 4.80;
piperazine), 3.63 (2H; s; –CH2–), 4.66 (2H; s; ClCH2–),
6.55 (1H; s; H5), 6.92–7.12 (4H; m; Ar–H), 9.05–9.20 ppm
(1H; br; –OH). ESI m/e 195 (100%), 353 (M ? 1, 38.32%),
355 (M ? 3), 375 (M ? 23).
¨ ˙
and Analyses Laboratory (TUBITAK-ATAL).
6-(Chloromethyl)-2-{[4-(4-fluorophenyl)piperazin-1-
yl]methyl}-3-hydroxy-4H-pyran-4-one (4)
2-Chloromethyl-5-hydroxy-4H-pyran-4-one (chlorokojic
acid) was synthesized as described before (Ellis et al.,
1996). Yield 60%, mp 166–167°C.
IR (KBr disc) 1623 (C=O st), 1510, 1461 (C=C st) and
1
1202 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
2.61 (4H; t; J = 4.80; piperazine), 3.07 (4H; t; J = 4.80;
piperazine), 3.63 (2H; s; –CH2–), 4.66 (2H; s; ClCH2–),
6.55 (1H; s; H5), 6.90–7.04 (4H; m; Ar–H), 9.05–9.20 ppm
(1H; br; –OH). ESI m/e 195 (100%), 353 (M ? 1, 20.35%),
355 (M ? 3), 375 (M ? 23).
General synthesis of Mannich base derivatives
(compounds 1–11)
Mannich bases were prepared by the reaction of substituted
phenyl piperazine derivatives (0.01 mol) and chlorokojic
acid (0.01 mol) in MeOH (15 ml) with 37% formaline
(1 ml). The mixture was stirred vigorously for 15–25 min
at room temperature. The resulting precipitate was col-
lected by filtration and washed with cold MeOH. The crude
product was recrystallized from MeOH except compound 9
which was recrystallized from CHCl3.
6-(Chloromethyl)-3-hydroxy-2-{[4-(2-
methoxyphenyl)piperazin-1-yl]methyl}-4H-pyran-4-one (5)
IR (KBr disc) 1623 (C=O st), 1499, 1452 (C=C st) and
1
1199 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
123

450
Med Chem Res (2011) 20:443–452
0
0
2.62 (4H; t; J = 4.40; piperazine), 2.96 (4H; t; J = 4.40;
piperazine), 3.62 (2H; s; –CH2–), 3.76 (3H; s; –OCH3),
4.67 (2H; s; ClCH2–), 6.55 (1H; s; H5), 6.87–6.94 ppm
(4H; m; Ar–H). ESI m/e 365 (M ? 1, 65.87%), 367
(M ? 3), 387 (M ? 23, 100%).
dd; J = 8.60, J = 2.20; Ar–H4 ), 6.92 (1H; s; Ar–H2 ), 7.19
0
(1H; t; J = 8.20; Ar–H5 ), 9.22–9.32 ppm (1H; br; –OH).
ESI m/e 195 (100%), 369 (M ? 1, 33.43%), 371 (M ? 3),
391 (M ? 23).
6-(Chloromethyl)-2-{[4-(4-chlorophenyl)piperazin-1-
yl]methyl}-3-hydroxy-4H-pyran-4-one (10)
6-(Chloromethyl)-3-hydroxy-2-{[4-(3-
methoxyphenyl)piperazin-1-yl]methyl}-4H-pyran-4-one (6)
IR (KBr disc) 1637 (C=O st), 1597, 1500 (C=C st) and
1
IR (KBr disc) 1623 (C=O st), 1497, 1455 (C=C st) and
1
1205 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
1202 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
2.50 (4H; t; J = 4.40; piperazine), 3.12 (4H; t; J = 4.40;
piperazine), 3.51 (2H; s; –CH2–), 4.55 (2H; s; ClCH2–),
2.61 (4H; t; J = 4.80; piperazine), 3.12 (4H; t; J = 4.80;
piperazine), 3.62 (2H; s; –CH2–), 3.70 (3H; s; –OCH3),
4.67 (2H; s; ClCH2–), 6.35 (1H; dd; J = 7.60, J = 2.40;
0
0
6.45 (1H; s; H5), 6.84 (2H; d; J = 8.95; Ar–H2 , H6 ), 7.10
0
0
(2H; d; J = 8.95; Ar–H3 , H5 ), 9.05–9.17 ppm (1H; br;
–OH). ESI m/e 101 (100%), 369 (M ? 1, 23.58%), 371
(M ? 3), 391 (M ? 23).
0
0
Ar–H2 ), 6.43 (1H; t; J =0 2.40; Ar–H4 ), 6.50 (1H; dd;
J = 8.40, J = 2.40; Ar–H6 ), 6.55 (1H; s; H5), 7.10 ppm
0
(1H; t; J = 8.20; Ar–H5 ), 9.01–9.30 ppm (1H; br; –OH).
ESI m/e 365 (M ? 1, 34.13%), 367 (M ? 3), 387
(M ? 23, 100%).
6-(Chloromethyl)-2-{[4-(3,4-dichlorophenyl)piperazin-1-
yl]methyl}-3-hydroxy-4H-pyran-4-one (11)
6-(Chloromethyl)-3-hydroxy-2-{[4-(4-
methoxyphenyl)piperazin-1-yl]methyl}-4H-pyran-4-one (7)
IR (KBr disc) 1630 (C=O st), 1596, 1487 (C=C st) and
1
1194 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
2.59 (4H; t; J = 4.80; piperazine), 3.18 (4H; t; J = 4.40;
piperazine), 3.62 (2H; s; –CH2–), 4.66 (2H; s; ClCH2–),
6.56 (1H; s; H5), 6.91 (1H; dd; J = 8.80, J = 2.80; Ar–
H6 ), 7.10 (1H; s; Ar–H2 ), 7.36 (1H; d; J = 8.80; Ar–H5 ),
9.22–9.32 ppm (1H; br; –OH). ESI m/e 101 (100%), 403
(M ? 1, 15.52%), 405 (M ? 3), 425 (M ? 23).
IR (KBr disc) 1625 (C=O st), 1514, 1458 (C=C st) and
1
1204 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
0
0
0
2.62 (4H; t; J = 5.00; piperazine), 3.00 (4H; t; J = 4.60;
piperazine), 3.62 (2H; s; –CH2–), 3.67 (3H; s; –OCH3),
4.66 (2H; s; ClCH2–), 6.55 (1H; s; H5), 6.80 (2H; d;
0
0
0
J = 7.80; Ar–H2 , H6 ), 6.86 (2H; d; J = 7.80; Ar–H3 ,
0
H5 ), 9.00–9.20 ppm (1H; br; –OH). ESI m/e 195 (100%),
365 (M ? 1, 83.83%), 367 (M ? 3), 387 (M ? 23).
Microbiological studies
6-(Chloromethyl)-2-{[4-(2-chlorophenyl)piperazin-1-
yl]methyl}-3-hydroxy-4H-pyran-4-one (8)
The compounds of 1–11 were dissolved in EtOH:hexane
(1:1) by using 1% Tween 80 solution at a final concen-
tration of 512 and 51.2 lg/ml and sterilized by filtration
using 0.22 lm Millipore (MA 01730, USA) and used as the
stock solutions. Reference antibacterial agents dissolved in
phosphate buffer solution (ampicillin, pH 8.0; 0.1 mol/ml),
dimethylsulphoxide (ketoconazole), or in water (gentami-
cin, ofloxacin, fluconazole) (NCCLS, 2006, 2008). Anti-
bacterial activity test were carried out against standards;
Gram-negative strains of E. coli ATCC 35218, P. aeru-
ginosa ATCC 10145, P. mirabilis ATCC 7002, K. pneu-
moniae RSKK 574, A. baumannii RSKK 02026, and as
Gram-positive strains of S. aureus ATCC 25923, E. fae-
calis ATCC 29212, B. subtilis ATCC 6633 and their drug
resistant isolates were used for the determination of anti-
bacterial activity. C. albicans ATCC 10231 and C. par-
apsilosis ATCC 22019 were used for the determination of
antifungal activity. Mueller-Hinton Broth (MHB; Difco)
and Mueller-Hinton Agar (MHA; Oxoid) were applied for
IR (KBr disc) 1622 (C=O st), 1479, 1454 (C=C st) and
1
1199 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
2.66 (4H; t; J = 4.40; piperazine), 2.98 (4H; t; J = 4.40;
piperazine), 3.65 (2H; s; –CH2–), 4.68 (2H; s; ClCH2–),
6.57 (1H; s; H5), 7.03 (1H; t; J = 7.80; Ar–H5 ), 7.14 (1H;
0
0
dd; J = 8.00, J = 1.20; Ar–H3 ), 7.28 (1H; t; J = 7.60;
0
0
Ar–H4 ), 7.39 (1H; dd; J = 7.80, J = 1.40; Ar–H6 ),
9.15–9.30 ppm (1H; br; –OH). ESI m/e 195 (100%), 369
(M ? 1, 30.75%), 371 (M ? 3), 391 (M ? 23).
6-(Chloromethyl)-2-{[4-(3-chlorophenyl)piperazin-1-
yl]methyl}-3-hydroxy-4H-pyran-4-one (9)
IR (KBr disc) 1630 (C=O st), 1454, 1429 (C=C st) and
1
1218 cm-1 (C–O st). H NMR d (DMSO-d6, 400 MHz)
2.59 (4H; t; J = 4.80; piperazine), 3.16 (4H; t; J = 4.40;
¨
growing and diluting of the bacteria suspensions (Ozc¸elik
piperazine), 3.62 (2H; s; –CH2–), 4.68 (2H; s; ClCH2–),
6.57 (1H; s; H5), 6.77 (1H; d; J = 7.60; Ar–H6 ), 6.88 (1H;
0
et al., 2006). The synthetic medium RPMI-1640 with
123

Med Chem Res (2011) 20:443–452
451
Brtko J, Rondahl L, Fickova M, Hudecova D, Eybl V, Uher M (2004)
Kojic acid and its derivatives: history and present state of art.
Cent Eur J Public Health 12:16–18
Burdock GA, Soni M, Carabin IG (2001) Evaluation of health aspects
of kojic acid in food. Regul Toxicol Pharmacol 33:80–101
Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS)
(2006) National Committee for Clinical Laboratory Standards.
Performance standards for antimicrobial susceptibility testing,
16th informational supplement. CLSI document M7-A7, 940
West Valley Road, Wayne, Pennsylvania 19087
Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS)
(2008) Reference method for broth dilution antifungal suscep-
tibility testing of yeast; approved standard, 3rd edn. CLSI
document M27-A3, Clinical and Laboratory Standards Institute,
940 West Valley Road, Suite 1400, Wayne, Pennsylvania, USA
Dehkordi LS, Liu ZD, Hider RC (2008) Basic 3-hydroxypyridine-4-
ones: potential antimalarial agents. Eur J Med Chem 43:1035–
1047
Ellis BL, Duhme AK, Hider RC, Hossain MB, Rizvi S, Van der Helm
D (1996) Synthesis, physicochemical properties, and biological
evaluation of hydroxypyranone and hydroxypyridinones: novel
bidentate ligands for cell-labeling. J Med Chem 39:3659–3670
Esquenazi D, Wigg MD, Miranda MFS (2002) Antimicrobial and
antiviral activities of polyphenolics from Cocos nucifera Linn.
(Palmae) husk fiber extract. Res Microbiol 153:647–652
Fassihi A, Abedi D, Saghaie L, Sabet R, Fazeli H, Bostaki G, Deilami
O, Sadinpour H (2009) Synthesis, antimicrobial evaluation and
QSAR study of some 3-hydroxypyridine-4-one and 3-hydrox-
ypyran-4-one. Eur J Med Chem 44(5):2145–2157
Foroumadi A, Ghodsi S, Emami S, Najjari S, Samadi N, Faramarzi
MA, Beikmohammadi L, Shirazi FH, Shafiee A (2006) Synthesis
and antibacterial activity of new fluoroquinolones containing a
substituted N-(phenethyl)piperazine moiety. Bioorg Med Chem
Lett 16:3499–3503
Hider RC, Singh S, Porter JB (1992) Iron chelating agents with
clinical potential. Proc R Soc Edinb 99B(1/2):137–168
Higa Y, Kawawbe M, Nabae K, Toda Y, Kitamoto S, Hara T, Tanaka
N, Kariya K, Takahashi M (2007) Kojic acid-absence of tumor-
initiating activity in rat liver, and of carcinogenic and photo-
genotoxic potential in mouse skin. J Toxicol Sci 32(2):143–159
Ichimoto I, Tatsumi C (1965) Studies on kojic acid and its related
c-pyrone compounds. Part VII. The alkylation of kojic acid and
pyromeconic acid through their Mannich base. Agric Biol Chem
1(2):94–98
L-glutamine was buffered to pH 7 with 3-[N-morpholino]-
propansulfonic acid and culture suspensions were prepared
¨
as described previously (Ozc¸elik et al., 2009). The mic-
rodilution method was employed for antibacterial and
antifungal activity tests. Media were placed into each 96
wells of the microplates. Extract solutions at 512 lg/ml
were added into first rows of microplates and twofold
dilutions of the compounds (256–0.125 lg/ml) were made
by dispensing the solutions to the remaining wells. The
lowest concentration of the extracts that completely inhibit
macroscopic growth was determined and minimum inhib-
itory concentrations (MICs) were reported as described
¨
previously study (Ozc¸elik et al., 2009).
Cytotoxicity and antiviral activity tests
Vero cell line (African green monkey kidney) used in this
study was obtained from Department of Virology, Faculty
of Veterinary, Ankara University (Ankara-Turkey). In
order to determine the antiviral activity of the extracts,
Herpes simplex virus Type-1 (HSV-1), as representative of
DNA viruses and Parainfluenza-3 virus (PI-3), as repre-
sentative of RNA viruses, were used. Media (EMEM) were
placed into each 96 wells of the microplates (GreinerÒ;
Essen, Germany). Stock solutions of the extracts were
added into first raws of microplates and twofold dilutions
of the extracts (51.2–0.012 lg/ml) were made by dis-
pensing the solutions to the remaining wells. The MNTCs
of each samples were determined by the method described
¨
previously study (Ozc¸elik et al., 2009) based on cellular
morphologic alteration.
Acknowledgments The authors wish to thank Taner Karaoglu,
Ph.D. for his kind help to conduct antiviral tests. This research is
´
funded as a project by L’Oreal Tu¨rkiye Fellowships for Young
Women in Science supported by The Turkish Academy of Sciences.
Kayahara H, Shibata N, Tadasa K, Maeda H, Kotani T, Ichimoto I
(1990) Amino acid and peptide derivatives of kojic acid and their
antifungal properties. Agric Biol Chem 54(9):2441–2442
Kotani T, Ichimoto I, Tatsumi C, Fujita T (1975) Structure–activity
study of bacteriostatic kojic acid analogs. Agric Biol Chem
39(6):1311–1317
References
Alverson J (2003) Effects of mycotoxins, kojic acid and oxalic acid,
on biological fitness of Lygus hesperus (Heteroptera: Miridae).
J Invertebr Pathol 83:60–62
O’brien GJ, Patterson M, Meadow JR (1960) Amino derivatives of
kojic acid. J Org Chem 25:86–89
¨
Aytemir MD, Erol DD, Hider RC, Ozalp M (2003) Synthesis and
¨
Ozc¸elik B, Orhan I, Toker G (2006) Antiviral and antimicrobial
evaluation of antimicrobial activity of new 3-hydroxy-6-methyl-
J Chem
assessment of some selected flavonoids. Z Naturforsch C 61:
632–638
Ozc¸elik B, Gu
¨rbu¨z I, Karaoglu T (2009) Antiviral and antimicrobial
4-oxo-4H-pyran-2-carboxamide derivatives. Turk
27(6):757–764
¨
¨
¨
activities of three sesquiterpene lactones from Centaurea
Aytemir MD, C¸ alıs¸ U, Ozalp M (2004) Synthesis and evaluation of
anticonvulsant and antimicrobial activities of 3-hydroxy-6-
methyl-2-substituted 4H-pyran-4-one derivatives. Arch Pharm
337:281–288
solstitialis L. ssp. soldtitialis. Microbiol Res 164(5):545–552
Pirselova K, Balaz S, Ujhelyova R, Sturdik E (1996) Quantitative
structure–time–activity relationships (QSTAR): growth inhibi-
tion of Escherichia coli by nonionizable kojic acid derivatives.
Quant Struct-Act Relat 15:87–93
¨
˘
Aytemir MD, Septioglu E, C¸ alıs¸ U (2010) Synthesis and anticonvul-
sant activity of new kojic acid derivatives. Arzneim-Forsch/Drug
Res 60(1):22–29
Bentley R (2006) From miso, sake and shoyu to cosmetics: a century
of science for kojic acid. Nat Prod Rep 23:1046–1062
ˇ
´
´
´
ˇ ´ˇ
Synytsya A, Blafkova P, Synytsya A, Copıkova J, Spevacek J, Uher
M (2008) Conjugation of kojic acid with chitosan. Carbohydr
Polym 72:21–31
´
123

452
Med Chem Res (2011) 20:443–452
Thomas SP, Patterson F (2002) Antifungal resistance in pathogenic
fungi. Clin Infect Dis 35:1073–1080
Weinberg ED (1957) The mutual effects of antimicrobial compounds
and metallic cations. Microbiol Mol Biol Rev 21:46–63
Wolf PA, Westveer WM (1950) The antimicrobial activity of several
substituted pyrones. Arch Biochem 28:201–206
Xiong X, Pirrung MC (2008) Modular synthesis of candidate indole-
based insulin mimics by Claisen rearrangement. Org Lett
10(6):1151–1154
Uchino K, Nagawa M, Tanasaki Y, Oda M, Fukuchi A (1988) Kojic
acid as an anti-speck agent. Agric Biol Chem 52:2609–2610
Uher M, Szymonska J, Korenova A, Tomasik P (2000) Re-exami-
nation of nucleophilic substitution in chlorokojic acid. Monatsh
Chem 131:301–307
Upadhayaya RS, Sinha N, Jain S, Kishore N, Chandra R, Arora SK
(2004) Optically active antifungal azoles: synthesis and anti-
fungal activity of (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-
aryl-piperazin-1-yl]-ethyl}-tetrazol-2-yl/1-yl)-1-[1,2,4]-triazol-
1-yl-butan-2-ol. Bioorg Med Chem 12:2225–2238
Zborowski K, Korenova A, Uher M, Proniewicz LM (2004) Quantum
chemical studies on tautomeric equilibria in chlorokojic and
azidokojic acids. J Mol Struct 683:15–22
Veverka M, Kralovicova E (1990) Synthesis of some biologically
active derivatives of 2-hydroxymethyl-5-hydroxy-4H-pyran-4-
one. Collect Czech Chem Commun 55:833–840
123