A study of cytotoxicity of novel chlorokojic acid derivatives with their antimicrobial and antiviral activities

Article abstract of DOI:10.1016/j.ejmech.2010.05.069

A series of 6-chloromethyl-3-hydroxy-2-substituted 4H-pyran-4-one derivatives were synthesized and tested for their antimicrobial and antiviral activities. Mannich base derivatives were prepared through the reaction of substituted piperazine or piperidine derivatives on chlorokojic acid and formaline. The structures of the synthesized compounds were confirmed by IR, ¹H and ¹³C NMR, ESI-MS, and elemental analysis. According to the activity studies, compounds 2-7 (MIC: 1-2 μg/mL) were found to be highly active against Bacillus subtilis and Staphylococcus aureus, while compounds 3, 5 and 6 showed significant activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter baumannii. Also, compounds 2-7 were more remarkably active against Candida albicans and Candida parapsilosis (MIC: 4-8 μg/mL). Additionally, compound 2 was the most active one against RNA virus PI-3. Seven novel 6-chloromethyl-3-hydroxy-2-substituted 4H-pyran-4-one derivatives (compounds 1-7) were synthesized and tested for their antimicrobial and antiviral activities.

Full text of DOI:10.1016/j.ejmech.2010.05.069

European Journal of Medicinal Chemistry 45 (2010) 4089e4095
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A study of cytotoxicity of novel chlorokojic acid derivatives with their
antimicrobial and antiviral activitiesq
,
b
Mutlu Dilsiz Aytemir a , Berrin Özçelik
*
a Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06100 Sıhhiye e Ankara, Turkey
b Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Microbiology, 06330 Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 6-chloromethyl-3-hydroxy-2-substituted 4H-pyran-4-one derivatives were synthesized and
tested for their antimicrobial and antiviral activities. Mannich base derivatives were prepared through
the reaction of substituted piperazine or piperidine derivatives on chlorokojic acid and formaline. The
structures of the synthesized compounds were confirmed by IR, 1H and 13C NMR, ESI-MS, and elemental
Received 22 February 2010
Received in revised form
12 May 2010
Accepted 29 May 2010
Available online 8 June 2010
analysis. According to the activity studies, compounds 2e7 (MIC: 1e2 mg/mL) were found to be highly
active against Bacillus subtilis and Staphylococcus aureus, while compounds 3, 5 and 6 showed significant
activity against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae and Acinetobacter bau-
mannii. Also, compounds 2e7 were more remarkably active against Candida albicans and Candida par-
Keywords:
Chlorokojic acid
Kojic acid
Mannich bases
Synthesis
apsilosis (MIC: 4e8
m
g/mL). Additionally, compound 2 was the most active one against RNA virus PI-3.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Antimicrobial
Antiviral
1. Introduction
resistance. Virally encoded drug resistance has been documented
against nearly all compounds with antiviral activity, and the genetic
In the last few decades, the frequency and spectrum of antimi-
crobial-resistant infections have increased both in the hospitals and
community. It is reported that certain infections that are essentially
untreatable have begun to occur as epidemics both in the devel-
oping and other developed regions as a result of antimicrobial
resistance. The most serious concern with antibiotic resistance is
that some bacteria have become resistant to almost all of the
readily available antibiotics and these bacteria can cause serious
diseases. Examples include methicillin-resistant Staphylococcus
aureus (MRSA), vancomycin-resistant Enterococcus (VRE) and
basis of resistance is known now [2]. Many screening efforts have
been made to find new antimicrobial or antiviral agents from
natural or synthetic compounds that can specifically act on
different molecular targets to control infections caused by various
microorganisms [3e7].
Wolf and Westveer showed that 2-chloromethyl-5-hydroxy-4H-
pyran-4-one (chlorokojic acid) contains catechol group-inhibited
Aeromonas aeruginosa, Micrococcus pyogenes var. aureus, Salmonella
typhosa, Penicillium digitatum, Russula nigricans and Saccharomyces
cerevisiae [8]. Kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-
one) and its derivatives also have an inhibitory effect on the growth
of E. coli and S. aureus [9,10]. Previous antimicrobial activity studies
had shown that kojic acid was more active against Gram-negative
bacteria than against Gram-positive ones [11]. However, some of its
derivatives have shown adverse effects different from kojic acid’s
antibacterial activity results [12e15]. Also, chlorokojic acid and
other halogen derivatives have significant antifungal activity.
Moreover, their copper(II) salts’ complex derivatives were prepared
and found to be more active than chlorokojic acid [16]. Kojic acid
and its derivatives exhibit a number of interesting bioactivities and
are used in food additives [11,16,17], herbicidals [12,18], anti-speck
agents [19], pesticides and insecticides [20e22], and also as a skin-
whitening product in cosmetic products [11,17].
multidrug-resistant Streptococcus pyogenes (Group
A Strepto-
coccus: GAS) [1]. This is a major public health issue. On the other
hand, the development of effective antiviral drugs is an important
biomedical scientific achievement of the late 20th century. These
are highly potent drugs against herpes viruses, human immuno-
deficiency virus (HIV), hepatitis B virus, influenza virus and with
extension of the list to papilloma viruses, respiratory viruses,
enteroviruses, and hepatitis C virus over the next 5e10 years. But
this exciting background comes with the problem of drug
q
The authors have declared no conflict of interest.
* Corresponding author. Tel.: þ90 312 324 03 33x114; fax: þ 90 312 311 4777.
E-mail address: mutlud@hacettepe.edu.tr (M.D. Aytemir).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.069

4090
M.D. Aytemir, B. Özçelik / European Journal of Medicinal Chemistry 45 (2010) 4089e4095
Fig. 1. Hydroxypyranone derivatives.
Previously, some of the Mannich bases of allomaltol (5-hydroxy-
a
Mannich group [27]. Additionally, 6-morpholino or piper-
2-methyl-4H-pyran-4-one) derivatives were prepared in our labo-
ratory for their anticonvulsant and antimicrobial activities. All these
derivatives have a lower antifungal activity than chlorokojic acid
[23]. Therefore, in order to continue our research program, seven
novel Mannich bases of chlorokojic acid derivatives were synthe-
sized and screened for their antimicrobial and antiviral activities.
idinomethyl chlorokojic acid was prepared via Mannich reaction
[30]. In a later study, Ichimoto et al. synthesized Mannich bases of
kojic acid and pyromeconic acid in either an acidic or a basic
medium using aliphatic or heterocyclic secondary amines such as
dimethylamine, diethylamine or morpholine, respectively [24].
Within the scope of the present study, using the methodology
shown in Scheme 1, seven new 6-chloromethyl-3-hydroxy-2-
substituted 4H-pyran-4-one derivatives were synthesized as
Mannich base derivatives. The basic substituent was introduced in
the 6th-position of chlorokojic acid via a Mannich-type reaction,
using formaline and an appropriate substituted piperidine or
piperazine derivatives in methanol at room temperature. The
reaction proceeded very rapidly. Formation of the desired new
Mannich base derivatives was confirmed on the basis of elemental
analysis, and the structures of the compounds were supported by
spectral data. The IR, 1H and 13C NMR, and ESI-MS were in agree-
ment with the proposed structures. Yields and the melting points of
the synthesized compounds are presented in Table 1. In the IR
spectra of all compounds, the stretching (st) bands associated with
C]O (pyranone), C]C and CeO were observed at 1657e1622,
1597e1456, and 1227e1198 cmꢀ1, respectively. With 1H NMR
spectra, characteristic singlet peaks of the 4H-pyran-4-one (H5)
ring proton were found in the region 6.53e6.56 ppm in accordance
with literatures [13,23,28,31]. The methylene group (ClCH2e)
proton signals were displayed as singlet peaks at 4.63e4.66 ppm.
The other eCH2e group proton signals of 1e7 appeared also as
singlet peaks at 3.43e3.62 ppm. 13C NMR spectra of compounds
1e7 had typical peaks for 6-chloromethyl and carbonyl of pyranone
ring were observed in the range of 41.33e42.10 and
173.36e174.2 ppm, respectively. Compounds 1 and 6 bearing fluoro
atoms showed suitable spinespin decoupling. All these results
were found acceptable with literature [23]. The ESI-MS of all
compounds showed (Mþ þ H), (Mþ þ Hþ2) and (Mþ þ Na) peaks.
2. Results and discussion
2.1. Chemistry
Kojic acid provides a promising skeleton for development of
new and more potent derivatives such as chlorokojic acid, allo-
maltol and pyromeconic acid (3-hydroxy-4H-pyran-4-one) (Fig. 1).
They are good ligands for the nucleophilic and electrophilic
substitution reactions [24e26]. Therefore, many researchers used
them as a starting or intermediate compound for the preparation of
new compounds in medicinal chemistry [13,20,23,27e31].
Chlorokojic acid was synthesized from commercially available
kojic acid in a one-step reaction as suggested in previous studies
[13,23,27,28]. Chlorination of the 2-hydroxymethyl moiety of kojic
acid using thionyl chloride at room temperature produced chlor-
okojic acid, with the ring hydroxyl being unaffected. Mannich-type
reactions are three-component condensation reactions involving
carbonyl compounds, existing as ketoeenol tautomeric forms,
formaline, and a primary or secondary amine. Because of its
phenol-like properties, kojic acid readily undergoes amino-
methylation at room temperature during the Mannich reaction
ortho to the enolic hydroxyl group. Woods has reported di-Mannich
derivatives obtained in an acidic medium from kojic acid, formaline
and aromatic amine [30]. Using dimethylamine, diethylamine,
pyrrolidine, morpholine, piperidine or 4-methylpiperazine, and
chlorokojic acid, only 6th-position of kojic acid was substituted by
Scheme 1. Preparation of Mannich bases. Reagents and conditions a: SOCl2; b: MeOH.

M.D. Aytemir, B. Özçelik / European Journal of Medicinal Chemistry 45 (2010) 4089e4095
4091
Table 1
As given in Table 2, the antibacterial activity against Gram-
negative bacteria of the synthesized compounds 3, 5, and 6 bearing
Yields and melting points of the synthesized compounds (1e7).
(4-chlorophenyl)benzylpiperazine,
4-bromophenyl-4-hydrox-
ypiperidine and 4-chloro-3-(trifluoromethyl)phenyl-4-hydrox-
ypiperidine moiety at the 2nd-position of pyran-4-one ring, was
found to have significantly high antibacterial potential against
standard strains of E. coli, P. aeruginosa, K. pneumoniae, A. baumannii
with a bacterial inhibition between 2 and 4
compounds showed more activity (MIC: 2 g/mL) against E. coli and
P. aeruginosa than the other Gram-negative bacteria compared with
control drugs ampicillin (MIC: 2 g/mL), ofloxacin (1 g/mL) and
levofloxacin (1 g/mL). The least efficiency of the compounds
among Gram-negative bacteria was seen on P. mirabilis with
a concentration range of 8e16 g/mL in comparison with ofloxacin
and levofloxacin (MICs: <0.12e1 g/mL).
mg/mL. Also, these
m
Compounds
1
R
M.p. (ꢂC)
Yield (%)
68
m
m
m
157e8
m
m
As for compounds 2 and 4, it was found that they had the same
effect against all Gram-negative bacteria. The MIC values of both
2
3
147e8
98e9
86
78
compounds were 4
coli, K. pneumoniae, and A. baumannii; and 16
mirabilis. Furthermore, on comparison, compound 7 had similar
mg/mL against P. aeruginosa; 8
mg/mL against E.
m
g/mL against P.
antibacterial activity with MIC values between 4 and 16 mg/mL as
compounds 2 and 4.
In the entire series, compound 1 possessing 4-fluorobenzyl
piperazine moiety was less effective (MIC: 16e32 g/mL) towards
standard strains of all Gram-negative bacteria besides compound 7
(16 g/mL), which showed the same activity on P. mirabilis.
Multiple drug-resistant organisms used in the current study are
becoming common causes of infections in the acute and long-term
care units of hospitals. The emergence of resistance to the beta-
lactam antibiotics has become more serious in recent decades.
Broad use of antibiotics is followed by emergence of resistant
m
4
5
6
7
143e4
138e9
122e3
158e9
80
43
50
82
m
strains such as extended-spectrum beta-lactamases (ESbLs). E. coli
and also A. baumannii are the opportunistic nosocomial pathogens
associated with significant morbidity and mortality. Additionally,
among drug-resistant microorganisms from highly prevalent
opportunistic pathogens, P. aeruginosa is responsible for 16% of
nosocomial pneumonia cases, 12% of hospital-acquired urinary
tract infections, 8% of surgical wound infections, and 10% of
bloodstream infections [1,3].
As shown in Table 2, the synthesized compounds revealed
a broad spectrum of activity with MIC values of 4e64 mg/mL against
tested drug-resistant isolated strains. Especially, compound 2
which has 3,4-dichlorobenzylpiperazine moiety showed remark-
2.2. In vitro antibacterial and antifungal activities
able activity against ES
with MIC values of 4 g/mL, compared with the reference drugs,
ampicillin (MIC: ꢁ128 g/mL), ofloxacin (MIC: 0.5 g/mL) and
levofloxacin (MIC: 0.5e1 g/mL, respectively). The other
compounds (1, 3e7) were found to manifest a moderate effect
(MICs: 16e64 mg/mL) for all isolated drug-resistant Gram-negative
strains.
As for structureeactivity relationship (SAR), fluoro substitution
in para position of the benzyl ring (compound 1) has the worst
b
L(þ) strains of E. coli and K. pneumoniae
m
The antibacterial and antifungal activity profiles of the newly
synthesized compounds were assessed for antimicrobial activity
against both the standard and the isolated strains of bacteria. For
antibacterial activity assessment, standard strains (Escherichia coli,
Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae,
Acinetobacter baumannii, S. aureus, Enterococcus faecalis and Bacillus
subtilis) and their drug-resistant isolates were tested; and for
antifungal activity Candida albicans and C. parapsilosis were used.
Ampicillin, vancomycin, gentamicin, ofloxacin, levofloxacin, keto-
conazole, and fluconazole were also tested under identical condi-
tions for comparison in antibacterial and antifungal assays,
respectively. Tables 2 and 3 describe the in vitro antimicrobial
activity with MIC values of compounds 1e7.
m
m
m
activity with MIC values between 16 and 32
whereas the 3,4-dichloro substitution (compound 2) of benzyl ring
increases antibacterial activity with four-folds (MIC: 4e8 g/mL)
towards isolated strains of A. baumannii and P. aeruginosa and two-
folds (MIC: 8 g/mL) against E. coli and K. pneumoniae. Moreover,
mg/mL in this series,
m
m
antibacterial activity of the compounds 1 and 2 against P. mirabilis
was determined to be the same. Furthermore, compounds 2 and 4
containing ester moiety had shown same activity against all
bacteria except from P. aeruginosa. In the Mannich base derivatives
bearing piperazine ring, compound 3 was the most remarkable and
According to our data (Tables 2 and 3), the synthesized
compounds showed a broad spectrum of activity against Gram-
positive and Gram-negative standard strains with MIC values
between 1 and 64
mg/mL. In the meantime, the synthesized
compounds showed activity against drug-resistant isolates of both
Gram-positive and -negative strains with MIC values of 2 to
active one (MIC: 2e8 mg/mL). It has two phenyl rings in its struc-
ture, of which one of them was a p-chlorophenyl ring and the other
ꢁ128
mg/mL.
a nonsubstituted phenyl ring. When compared with compounds 2

4092
M.D. Aytemir, B. Özçelik / European Journal of Medicinal Chemistry 45 (2010) 4089e4095
Table 2
Antibacterial activity of the synthesized compounds (1e7) and the control drugs (MIC in mg/mL).
Compounds Gram-negative standard and clinical isolated strains
E. coli
P. aeruginosa
P. mirabilis
K. pneumoniae
A. baumannii
ATCC 35218 Isolated strain ATCC 10145 Isolated strain ATCC 7002 Isolated strain RSKK 574 Isolated strain RSKK 02026 Isolated strain
1
2
3
4
5
6
7
AMP
GM
OFX
LVX
16
8
4
8
4
4
8
2
64
4
64
64
64
64
64
>128
e
16
4
2
4
2
64
16
16
16
16
16
16
e
16
16
8
16
8
8
16
2
64
64
64
64
64
64
64
>128
e
16
8
4
8
4
4
8
2
64
4
64
64
64
64
64
>128
e
32
8
4
8
4
4
4
2
e
64
16
16
16
16
16
16
>128
e
2
4
e
e
0.5
1
1
2
64
64
e
e
0.12
<0.12
0.5
0.5
<0.12
<0.12
1
1
<0.12
<0.12
0.5
1
0.12
0.12
64
64
AMP: ampicillin; GM: gentamicin; OFX: ofloxacin; LVX: levofloxacin, E. coli isolates (þES
bL enzyme), P. aeruginosa isolates (resist to Trimethoprim-Sulfamethoxazole,
L enzyme), A. baumannii isolates (resist to cephalosporin) -: No activity observed.
tazobactam), P. mirabilis isolates (þES
b
L enzyme), K. pneumoniae; isolates (þES
b
and 4, the addition of phenyl ring on the structure of compound 3
increased the activity two-folds against all Gram-negative bacteria
and S. aureus. When antibacterial activity of compounds 5e7 pos-
sessing piperidine ring was investigated, it was observed that
compounds 5 and 6 were found to have the same activity and
higher effect than compound 7 without halogen substitution at its
structure. Hence, when hydroxy substitution at the 4th-position of
piperidine ring was changed with acetyl, antibacterial activity was
decreased. In addition, there was no difference in the antimicrobial
activity with the location and type of the halogen substituted on
phenyl ring. Also, the compounds (5 and 6) had exactly the same
activity as compound 3 possessing piperazine ring against all
Gram-negative bacteria.
Candida species are the most widespread and threatening fungal
pathogens today, and are responsible for most of the invasive and
non-invasive fungal infections. Among all Candida species, C. albi-
cans is the most frequent pathogen [3,4].
The results obtained clearly indicate that the series of Mannich
bases discussed here are active towards growth inhibition of
pathogenic fungi. In general, all compounds, except compound 1,
exhibited excellent antifungal activity (MIC: 4e8
Candida spp when compared to the reference drugs, ketoconazole
(MIC: 1 g/mL) and fluconazole (MIC: 2e4 g/mL). The compounds
mg/mL) against
m
m
2e7 may be promoted as fungicides. In general, the compounds
showed an improved antibacterial activity when compared to their
antifungal activity.
Among Gram-positive bacteria, S. aureus has been recognized for
long as one of the major resistant pathogens that can cause diseases
in humans. Likewise, multi-drug resistant Enterococci have become
a serious threat for public health. High level resistance for penicillin
and aminoglycosides are being reported of this bacterium [32,33].
According to the obtained data (Table 3), antibacterial activity
2.3. Antiviral activity
All compounds were assayed against both herpes simplex virus-
1 (HSV-1) and human parainfluenza virus type 3 (PI-3) by using
Madin Darby Bovine Kidney and Vero cell lines with the aim to
capture structure relationship in each of the compounds. Acyclovir
and oseltamivir were used as control agents. Correlation between
toxicity on uninfected cells (Vero, MDBK) and antiviral activity of
the synthesis compounds were determined in the same microtiter
plate. The results of the antiviral study are presented in Table 4.
HSV-1 is a DNA virus that causes cold sore, a recurrent labial
infection, as well as genital infections less frequently. Para-
influenza viruses are enveloped RNA viruses which are the major
results ofcompounds 2e7 (MIC: 1e2
positive bacteria were encouraging, although compound 1 was
found to manifest moderate (MIC: 32 g/mL) activity against stan-
mg/mL) against standard Gram-
m
dard strains of S. aureus. All the synthesized substituted piperazine
or piperidine derivatives, except compound 1, were found to be
highlyactiveagainst B. subtilis showing a bacterial inhibitionvalue at
1 mg/mL but all synthesized compounds were less effective against E.
faecalis than the other Gram-positive bacteria.
Table 3
Antibacterial and antifungal activities of the synthesized compounds (1e7) and the control drugs (MIC in mg/mL).
Compounds
Gram-positive standard and clinical isolated strains
Fungi
S. aureus
E. faecalis
B. subtilis
C. albicans
ATCC 10231
C. parapsilosis
ATCC 25923
Isolated strain
ATCC 29212
Isolated strain
ATCC 6633
Isolated strain
ATCC 22019
1
2
3
4
5
6
7
AMP
VAN
OFX
LVX
KET
FLU
32
2
1
2
1
64
128
64
128
64
16
16
16
8
16
16
64
0.5
e
32
128
32
128
32
32
1
1
1
1
64
2
2
2
2
16
4
4
4
4
16
8
8
8
8
1
2
64
32
1
1
2
2
4
4
8
8
128
>128
2
64
128
e
128
>128
e
<0.12
0.12
0.25
0.25
e
0.12
e
e
e
e
e
0.5
e
e
e
e
e
e
e
e
e
1
e
e
e
e
1
1
0.5
e
32
32
e
e
e
e
e
2
4
AMP: ampicillin; VAN: vancomycin; OFX: ofloxacin; LVX: levofloxacin, KET; ketoconazole; FLU: fluconazole, S. aureus isolates (meticillin resist; MRSA), E. faecalis isolates
(cephalosporin resist), B. subtilis isolates (ceftriaxon resist) -: No activity observed.

M.D. Aytemir, B. Özçelik / European Journal of Medicinal Chemistry 45 (2010) 4089e4095
4093
Table 4
4. Experimental procedure
Cytotoxicity on MDBK and Vero cells as well as antiviral activity against HSV-1 and
PI-3 results.
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 (Phila-
delphia, PA, USA) and were uncorrected. IR spectra were recorded
MDBK cells
Compounds
Vero cells
MNTCs
MNTCs
g/mL)
CPE inhibitory
concentration
CPE inhibitory
concentration
(m
(mg/mL)
HSV-1
PI-3
on a Jasco FT/IR-420 spectrometer as KBr disc (g
, cmꢀ1). 1H NMR
Max.
Min.
Max.
Min.
0.05
0.05
0.1
spectra were obtained with a Varian 400 MHz spectrophotom-
eter in d6-dimethylsulfoxide (DMSO-d6) and tetramethylsilane
1
2
3
4
5
6
7
1.6
0.4
0.4
0.8
0.4
0.8
0.4
1.6
e
0.4
e
0.1
e
0.4
0.8
0.4
0.4
0.8
0.4
0.4
e
0.4
0.8
0.2
0.4
0.2
0.2
0.4
e
(TMS) was used as an internal standard (chemical shift in
d,
e
e
ppm). 13C NMR spectra were recorded on a Varian Mercury 400
spectrophotometer. Mass analysis was carried out with a Micro-
mass ZQ LC-MS with Masslynx Software Version 4.1 by using ESI
(þ) method. The elemental analyses were performed with a Ele-
mentar Analysensysteme GmbH varioMICRO CHNS at The
Scientific & Technological Research Council of Turkey-Ankara
0.8
e
0.4
e
0.2
0.05
0.05
0.05
0.8
e
0.2
e
Asiklovir
Oseltamivir
1.6
e
<0.012
e
e
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.
_
Testing and Analyses Laboratory (TÜBITAK-ATAL). The purity of
the compounds was assessed by thin layer chromatography (TLC)
on Kieselgel 60 F254 (Merck, Darmstadt, Germany)
chromatoplates.
Chlorokojic acid was synthesized as described by Ellis et al. [28].
causative agents of respiratory viral infections, and carry signifi-
cant high rates of morbidity and mortality in infants and young
children [34].
As given in CPE inhibitory concentration ranging, compounds 1
and 6 showed anti-Herpes simplex activity with less potency.
Among the tested Mannich base derivatives, compounds 2, 3, 5 and
6 were not active against DNA virus. Taking into account the CPE
inhibitory concentration ranging against the RNA viruses PI-3,
compound 2 containing 3,4-dichlorobenzyl moiety (0.8e0.05 mg/
mL) had remarkable antiviral activity in Mannich base derivatives.
Thereto, compounds 1 and 7 were less active than compound 2.
While the activities of compounds 1 and 7 (0.4e0.05 mg/mL) against
PI-3 were in similar CPE inhibitory concentration range,
compounds 5 and 6 had lower activity than that of compounds 1
and 7. Also, the activities of compounds 3 and 4 were of negligible
values as seen in Table 4.
Investigation among maximum non-toxic concentrations
(MNTCs) of synthesized compounds 1e7 on the Vero and MDBK
Yield 60%, m.p. 166e167 ꢂC.
4.1. General synthesis of Mannich bases (compounds 1e7)
Mannich bases were prepared by the reaction of substituted
piperazine or piperidine 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 15e25 min at room tempera-
ture. The resulting precipitate was collected by filtration and
washed with cold methanol. All crude products recrystallized
from MeOH except compound 6 which used chloroform as
a solvent.
Seven compounds were synthesized according to the proce-
dures as shown below.
4.1.1. 6-Chloromethyl-2-[4-(4-fluorobenzyl)piperazin-1-ylmethyl]-
3-hydroxy-4H-pyran-4-one (1)
IR (KBr disc) 1629 (C]O st), 1508, 1458 (C]C st) and 1205 cmꢀ1
(CeO st). 1H NMR (400 MHz, DMSO-d6, in ppm): 2.36 (4H; br s;
piperazine), 2.50 (4H; br s; piperazine), 3.43 (2H; s; eCH2e), 3.56
(2H; s;]NeCH2e), 40.65 (2H; s; ClCH2e), 6.54 (1H; s; H5), 7.11 (2H;
cell line was found to be 0.4e1.6
compounds 2e7 (0.4e0.8 g/mL) were more toxic than
compound 1 (1.6 g/mL) on MDBK cell culture line. At this
mg/mL. As per this result,
m
m
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 concentrations.
0
0
0
t; J ¼ 9.0; Ph-H3 , H5 ), 7.30 (2H; t; J ¼ 7.0; Ph-H2 , H6 ), 9.00e9.20
(1H; br; eOH). 13C NMR (100 MHz, DMSO-d6, in ppm): 42.09, 52.98,
53.04, 54.09, 61.66, 113.11, 115.50 (d; J ¼ 20.6), 131.26 (d; J ¼ 7.6),
134.93, 144.73, 148.34, 161.82, 161.89 (d; J ¼ 240.8; CeF), 174.12. ESI-
MS m/z 195 (100%), 367 (MþþH), 369 (Mþ þ Hþ2), 389 (Mþ þ Na).
Anal. Cal. for C18H21ClN2O4 MW: 366.11 Cal. C: 58.94 H: 5.50 N: 7.64.
Found C: 58.83 H: 5.39 N: 7.56.
3. Conclusion
In this study, the impact of halogen substituent at piperazine
or piperidine moiety was investigated. Among these Mannich
base derivatives, compounds 3, 5, and 6 were the most active
compounds towards S. aureus and B. subtilis with MIC values of
4.1.2. 6-Chloromethyl-2-[(4-(3,4-dichlorobenzyl)piperazin-1-yl)
methyl]-3-hydroxy-4H-pyran-4-one (2)
IR (KBr disc) 1625 (C]O st), 1597, 1456 (C]C st) and 1227 cmꢀ1
(CeO st). 1H NMR (400 MHz, DMSO-d6, in ppm):2.37 (4H; br s;
piperazine), 2.50 (4H; br s; piperazine), 3.45 (2H; s; eCH2e), 3.56
1
m
g/mL and P. aeruginosa with MIC values of 2
tively. Antibacterial activities of compounds 2e7 with MIC
values of 2 g/mL were determined for isolated drug-resistant
mg/mL, respec-
m
(2H; s;]NeCH2e), 4.65 (2H; s; ClCH2e), 6.54 (1H; s;0H5), 7.28 (1H;
0
strains of B. subtilis. All the synthesized compounds had better
antibacterial effect against Gram-positive bacteria than Gram-
negative ones. According to antifungal potential, compounds 2e7
were determined to be the most remarkably active Mannich
base derivatives against C. albicans and C. parapsilosis. When the
synthesized compounds were compared within CPE inhibitory
concentration ranges, compound 2 appeared the most active
derivative against PI-3.
dd; J ¼ 8.0; J ¼ 1.6; Ph-H5 ), 7.51 (1H; d; J ¼ 1.6; Ph-H2 ), 7.55 (1H; d;
0
J ¼ 8.0; Ph-H6 ), 9.00e9.20 (1H; br; eOH). 13C NMR (100 MHz,
CDCl3, in ppm): 41.43, 52.96, 53.07, 55.59, 61.73, 112.55, 128.51,
130.42, 131.96, 131.22, 132.56, 138.66, 144.51, 146.23, 161.64, 173.89.
ESI-MS m/z 101 (100%), 417 (Mþ þ H), 419 (Mþ þ Hþ2), 439
(Mþ
þ
Na). C18H19Cl3N2O3 for MW: 416.04 Anal. Cal. for
C18H19Cl3N2O3.1/2H2O MW: 426.04 Cal. C: 50.66 H: 4.72 N: 6.56.
Found C: 50.88 H: 4.59 N: 6.54.

4094
M.D. Aytemir, B. Özçelik / European Journal of Medicinal Chemistry 45 (2010) 4089e4095
4.1.3. 6-Chloromethyl-2-{[4-((4-chlorophenyl)(phenyl)methyl)
piperazin-1-yl]methyl}-3-hydroxy-4H-pyran-4-one (3)
54.47, 113.12, 127.11, 127.70, 129.52, 141.97, 144.70, 148.46, 161.82,
174.09, 209.49. ESI-MS m/z 101 (100%), 376 (Mþ þ H), 378
(Mþ þ Hþ2), 398 (Mþ þ Na). Anal. Cal. for C20H22ClNO4 MW: 375.12
Cal. C: 63.91 H: 5.90 N: 3.73. Found C: 63.52 H: 5.91 N: 3.61.
IR (KBr disc) 1633 (C]O st), 1586, 1479 (C]C st) and 1200 cmꢀ1
(CeO st). 1H NMR (400 MHz, DMSO-d6, in ppm): 2.30 (4H; br s;
piperazine), 2.50 (4H; br s; piperazine), 3.56 (2H; s; eCH2e), 4.32
(1H; s; eCH]), 4.64 (2H; s; ClCH2e), 6.54 (1H; s; H5), 7.16e7.43
(9H; m; Ph). 13C NMR (100 MHz, CDCl3, in ppm): 41.43, 51.72, 53.41,
55.61, 75.42, 112.60, 127.51, 128.02, 128.89, 128.94, 129.37, 132.88,
141.26, 142.03, 144.53, 146.28, 161.64, 173.93. ESI-MS m/z 101
(100%), 459 (Mþ þ H), 461 (Mþ þ Hþ2), 481 (Mþ þ Na). Anal. Cal.
for C24H24Cl2N2O3 MW: 458.11 Cal. C: 61.54 H: 5.38 N: 5.98. Found
C: 61.65 H: 5.33 N: 5.90.
4.2. Microbiological studies
4.2.1. Test materials
The compounds 1e7 were dissolved in EtOH:hexane (1:1) by
using 1% Tween 80 solution at a final concentration of 512 and
51.2
mg/mL and sterilized by filtration using 0.22 mm Millipore
(MA 01730, USA) and used as the stock solutions. Ampicillin,
vancomycin, gentamicin, ofloxacin, levofloxacin, ketoconazole
and fluconazole were used as the standard antibacterial and
antifungal drugs. Reference antibacterial agents of ampicillin
(AMP, Faco), gentamicin (GM; Faco), ofloxacin (OFX, Hoechst
Marion Roussel) reference antifungal agents of ketoconazole
(KET, Bilim) and fluconazole (FLU, Pfizer), were obtained from
their respective manufacturers and dissolved in phosphate buffer
solution (ampicillin, pH 8.0; 0.1 mol/mL), dimethylsulphoxide
(ketoconazole), or in water (gentamicin, ofloxacin, fluconazole).
The stock solutions of the agents were prepared in medium
according to the Clinical and Laboratory Standards Institute
[35,36].
4.1.4. tert-Butyl 4-[(6-(chloromethyl)-3-hydroxy-4-oxo-4H-pyran-
2-yl)methyl]piperazine-1-carboxylate (4)
IR (KBr disc) 1698 (C]O st, ester) and 1622 (C]O st, pyranone),
1457 cmꢀ1 (C]C st) and 1207 cmꢀ1 (CeO st). 1H NMR (400 MHz,
DMSO-d6, in ppm): 1.38 (9H; s; eCH3), 2.41 (4H; t; J ¼ 4.80;
piperazine), 3.31 (4H; t; J ¼ 4.80; piperazine), 3.59 (2H; s; eCH2e),
4.65 (2H; s; ClCH2e), 6.54 (1H; s; H5), 9.10e9.35 (1H; br; eOH). 13
C
NMR (100 MHz, CDCl3, in ppm): 28.62, 41.43, 52.80, 52.90, 55.25,
80.12, 112.11, 144.28, 146.04, 154.81, 162.07, 173.79. ESI-MS m/z 101
(100%), 359 (Mþ þ H), 361 (Mþ þ Hþ2), 381 (Mþ þ Na). Anal. Cal.
for C16H23ClN2O5 MW: 358.12 Cal. C: 53.56 H: 6.46 N: 7.81. Found C:
53.47 H: 6.42 N: 7.72.
4.2.2. Microorganisms and inoculums preparation
4.1.5. 2-{[4-(4-Bromophenyl)-4-hydroxypiperidin-1-yl]methyl}-6-
chloromethyl-3-hydroxy-4H-pyran-4-one (5)
Antibacterial activity test was carried out against standard
(ATCC; American type culture collection, RSKK; Culture collection
of Refik Saydam Central Hygiene Institute) and isolated (clinical
isolate and obtained from Department of Microbiology, Faculty of
Medicine, Gazi University) strains.
As standards; Gram-negative strains of E. coli ATCC 35218, P.
aeruginosa ATCC 10145, P. mirabilis ATCC 7002, K. pneumoniae RSKK
574, A. baumannii RSKK 02026, and Gram-positive strains of S.
aureus ATCC 25923, E. faecalis ATCC 29212, B. subtilis ATCC 6633
were used for the determination of antibacterial activity. The iso-
lated strains of E. coli, P. mirabilis and K. pneumoniae have extended-
IR (KBr disc) 1655 (C]O st), 1585 (C]C st) and 1205 cmꢀ1 (CeO
st). 1H NMR (400 MHz, DMSO-d6, in ppm): 1.58 (2H; d; J ¼ 12.40;
piperidine), 1.91 (2H; t; J ¼ 12.60; piperidine), 2.59 (2H; t; J ¼ 10.80;
piperidine), 2.66 (2H; d; J ¼ 10.80; piperidine), 3.61 (2H; s; eCH2e),
4.66 (2H; s; ClCH2e), 6.55 (1H; s; H5), 7.43 (2H; d; J ¼ 8.80; Ph), 7.48
(2H; d; J ¼ 8.40; Ph). 13C NMR (100 MHz, DMSO-d6, in ppm): 37.61,
41.33, 48.49, 48.65, 53.79, 69.08,112.37,119.23,127.14,130.56,143.9,
147.90, 149.32, 161.05,173.36. ESI-MS m/z 101 (100%), 428 (Mþ þ H),
430 (Mþ þ Hþ2), 450 (Mþ þ Na). C18H20BrClNO4 for MW: 427.02
Anal. Cal. for C18H19BrClNO4.1/2H2O MW: 437.71 Cal. C: 49.39 H:
4.60 N: 3.19. Found C: 49.19 H: 4.95 N: 2.97.
spectrum
b-lactamase (ESbL) enzyme, P. aeruginosa isolate (resis-
tant toTrimethoprim-Sulfamethoxazole, tazobactam), A. baumannii
isolate (resistant to cephalosporin), Gram-positive isolated strains
of S. aureus (resistant to methicillin; MRSA), E. faecalis (resistant to
cephalosporin), B. subtilis (resistant to ceftriaxon) were used for the
determination of antibacterial activity. C. albicans ATCC 10231 and
C. parapsilosis ATCC 22019 were used for the determination of
antifungal activity.
MuellereHinton Broth (MHB: Difco) and MuellereHinton Agar
(MHA: Oxoid) were applied for growth and dilution of the bacterial
suspensions. The synthetic medium RPMI-1640 with L-glutamine
was buffered to pH 7 with 3-[N-morpholino]-propansulfonic acid
and culture suspensions were prepared. The microorganism
suspensions used for inoculation were prepared at 105 c.f.u. (colony
forming unite/mL) by diluting fresh cultures at McFarland 0.5
density (108 c.f.u./mL). Suspensions of bacteria and fungi were
added in each well of the diluted extracts with a density of 105 c.f.
u./mL for fungi and bacteria. The bacterial suspensions used for
inoculation were prepared at 105 c.f.u./mL by diluting fresh
cultures at McFarland 0.5 density (108 c.f.u./mL). The fungi
suspension was prepared by the spectrophotometric method of
inoculums [36].
4.1.6. 2-{[4-(4-Chloro-3-(trifluoromethyl)phenyl)-4-
hydroxypiperidin-1-yl]methyl}-6-chloromethyl-3-hydroxy-4H-
pyran-4-one (6)
IR (KBr disc) 1657 (C]O st), 1586, 1479 (C]C st) and 1198 cmꢀ1
(CeO st). 1H NMR (400 MHz, DMSO-d6, in ppm): 1.58 (2H; d;
J ¼ 12.00; piperidine), 1.97 (2H; t; J ¼ 12.60; piperidine), 2.59 (2H; t;
J ¼ 10.8; piperidine), 2.71 (2H; d; J ¼ 10.40; piperidine), 3.62 (2H; s;
eCH2e), 4.66 (2H; s; ClCH2e), 50.00e5.20 (1H; br; eOH), 6.56 (1H; s;
H5), 7.64 (1H; d; J ¼ 8.0; Ph-H3 ), 7.76 (1H; dd; J ¼ 8.8; J ¼ 2.0; Ph-
0
0
H2 ), 7.91 (1H; d; J ¼ 2.0; Ph-H6 ). 13C NMR (100 MHz, DMSO-d6, in
ppm): 38.20, 42.10, 49.30, 54.50, 69.98, 113.15, 122.36, 124.92,
126.73 (q; J ¼ 30.4; eCF3), 127.8, 129.2, 131.4, 131.9, 144.8, 148.6,
161.8, 174.2. ESI-MS m/z 101 (100%), 452 (Mþ þ H), 454 (Mþ þ Hþ2),
474 (Mþ
þ
Na). C19H18Cl2F3NO4 MW: 451.05 Anal. Cal. for
C19H18Cl2F3NO4.H2O MW: 469.06 Cal. C: 48.52 H: 4.28 N: 2.97.
Found C: 48.04 H: 4.38 N: 2.80.
4.1.7. 2-[(4-Acetyl-4-phenylpiperidin-1-yl)methyl]-6-
chloromethyl-3-hydroxy-4H-pyran-4-one (7)
IR (KBr disc) 1695 (C]O st, carbonyl), 1651 (C]O st, pyranone),
1586 (C]C st) and 1198 cmꢀ1 (CeO st). 1H NMR (400 MHz, DMSO-
d6, in ppm): 1.86 (3H; s; eCOCH3),1.97 (2H; d; J ¼ 11.40; piperidine),
2.03e2.65 (6H; m; piperidine), 3.51 (2H; s; eCH2e), 4.63 (2H; s;
ClCH2e), 6.53 (1H; s; H5), 7.25e7.99 (5H; m; Ph). 13C NMR
(100 MHz, DMSO-d6, in ppm): 26.10, 32.84, 42.07, 50.75, 54.21,
4.2.3. Antibacterial and antifungal tests
The microdilution method was employed for antibacterial and
antifungal activity tests. Media were placed into each 96 wells of
the microplates. Extract solutions at 512
first rows of microplates and two-fold dilutions of the compounds
mg/mL were added into

M.D. Aytemir, B. Özçelik / European Journal of Medicinal Chemistry 45 (2010) 4089e4095
4095
(256e0.125
mg/mL) were made by dispensing the solutions to the
Acknowledgments
remaining wells. 10
mL culture suspensions were inoculated into all
the wells. DMSO (80%) and EtOH (20%), pure microorganisms and
pure media were used as control wells. All organisms were tested in
triplicate in each run of the experiments. The sealed microplates
were incubated at 35 ꢂC for 24 h and 48 h in humid chamber. The
lowest concentration of the compounds that completely inhibit
macroscopic growth was determined and minimum inhibitory
concentrations (MICs) were reported [3,35]. The MICs were evalu-
ated on test samples that showed antimicrobial activity.
The authors wish to thank Taner Karaoglu, Ph.D. for his kind
help in conducting antiviral tests. This research is funded as
a project by L’Oréal Türkiye Fellowships for Young Women in
Science supported by The Turkish Academy of Sciences. The Central
Laboratory of the Faculty of Pharmacy of Ankara University
provided support for acquisition of the IR, 1H and 13C NMR spec-
trometer used in this work. We would like to thank Professor Erhan
Palaska for his help with the ESI-MS analysis.
4.2.4. Cytotoxicity and antiviral activity tests
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