Design, synthesis and in vivo/in vitro screening of novel chlorokojic acid derivatives

Article abstract of DOI:10.3109/14756366.2012.666538

A series of novel Mannich bases of chlorokojic acid (2-chloromethyl-5- hydroxy-4H-pyran-4-one) were synthesized and their biological activities were investigated. Anticonvulsant activity results according to phase-I tests of Antiepileptic Drug Development (ADD) Program revealed that compound 13 was the most effective one at 4h against subcutaneous pentylenetetrazole (scPTZ)-induced seizure test. Antimicrobial activities were evaluated in vitro against bacteria and fungi by using broth microdilution method. The antitubercular activities against Mycobacterium tuberculosis and M. avium were discussed with Resazurin microplate assay (REMA). The antimicrobial activity results indicated that compounds 1 and 12 (MIC: 8-16 g/mL) showed higher activity against Gram negative bacteria while compound 12 had MIC: 4-16 g/mL against Gram positive bacteria. Compound 1 was the most active one with MIC values of 8-32 g/mL against fungi. Mannich bases also exhibit significant antitubercular activity in a MIC range of 4 to 32 g/mL, especially compound 18 against M. avium.

Full text of DOI:10.3109/14756366.2012.666538

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; Early Online: 1–12
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.666538
RESEARCH ARtIClE
Design, synthesis and in vivo/in vitro screening of
novel chlorokojic acid derivatives
Gülşah Karakaya1, Mutlu Dilsiz Aytemir1, Berrin Özçelik2, and Ünsal Çalış1
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey and
2Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Gazi University, Ankara, Turkey
Abstract
A series of novel Mannich bases of chlorokojic acid (2-chloromethyl-5-hydroxy-4H-pyran-4-one) were synthesized
and their biological activities were investigated. Anticonvulsant activity results according to phase-I tests of
Antiepileptic Drug Development (ADD) Program revealed that compound 13 was the most effective one at 4h
against subcutaneous pentylenetetrazole (scPTZ)-induced seizure test. Antimicrobial activities were evaluated in vitro
against bacteria and fungi by using broth microdilution method. The antitubercular activities against Mycobacterium
tuberculosis and M. avium were discussed with Resazurin microplate assay (REMA). The antimicrobial activity results
indicated that compounds 1 and 12 (MIC: 8–16 µg/mL) showed higher activity against Gram negative bacteria while
compound 12 had MIC: 4–16 µg/mL against Gram positive bacteria. Compound 1 was the most active one with MIC
values of 8–32 µg/mL against fungi. Mannich bases also exhibit significant antitubercular activity in a MIC range of 4
to 32 µg/mL, especially compound 18 against M. avium.
Keywords: Chlorokojic acid, Mannich bases, anticonvulsant, antimicrobial, antimycobacterial
Introduction
and insecticide20, antitumor activity21 and anti-diabetic
agent22.
Kojic acid (5-hydroxy-2-hydroxymethyl-4H-pyran-4-one),
is a biologically important fungal metabolite, produced by
many species of Aspergillus, Acetobacter and Penicillium.
Becauseofprovidingapromisingskeletonfordevelopment
of both new and more potent derivatives, it has been used
by many researchers as a starting material in preparation
of new compounds1–11. Chlorokojic acid (2-chloromethyl-
5-hydroxy-4H-pyran-4-one), which is synthesized by
chlorination of kojic acid exhibits distinct antibacterial
and antifungal activities3–5. Moreover, these hydroxypy-
rone derivatives are known as effective chelating agents
forming complexes with metals5,12,13 and this ability plays
a significant role in antimicrobial activity14. Kojic acid and
its derivatives also display a variety of biological activities,
such as antiepileptic4,6–9, modest anti-inflammatory agent5,
food additive15, antioxidant or antibrowning agent16, skin-
lightening product in cosmetics as a result of inhibition of
melanin production17, herbicidal18, anti-speck19, pesticide
Epilepsy, one of the more common neurological dis-
orders, affects a large section of people. Since available
marketed antiepileptic drugs possess the risk of tolerance
development, dose-related toxicity, and idiosyncratic
side effects, the search for a new agent is still a popular
investigation area of medicinal chemists worldwide23. In
the literature many compounds bearing hydroxypyrone
ring such as kojic acid, kojic amine, maltol and etil maltol
have been reported as anticonvulsant agents24–27. e
lipid solubility of a drug is an important factor in con-
nection with its transfer into the central nervous system.
e increase of anticonvulsant effect in Mannich bases
of kojic acid and allomaltol (5-hydroxy-2-methyl-4H-
pyran-4-one) is attributed to increase of lipophilicity4,8,9
.
Tuberculosis (TB), one of the earliest recorded human
diseases and a chronic bacterial infection, continues to
be an important global one with mortality worldwide kill-
ing more than 2 million people a year28,29. Mycobacterium
Address for Correspondence: Prof. Dr. Mutlu D. Aytemir, Hacettepe University, Faculty of Pharmacy, Department of Pharmaceutical
Chemistry, 06100 Sıhhiye, Ankara, Turkey. Tel: + 90 312 324 03 33-114; Fax: + 90 312 311 4777. E-mail: mutlud@hacettepe.edu.tr
(Received 05 December 2011; revised 08 February 2012; accepted 08 February 2012)
1

2
G. Karakaya et al.
tuberculosis, which causes TB, infects approximately
one-third of the world’s population and is comparable
only to human immunodeficiency virus (HIV) as an
infectious cause of death30. Also, Mycobacterium avium
complex (MAC) is the most common cause of human
infection due to nontuberculous mycobacteria. MAC is
not only an opportunistic pathogen in acquired immu-
nodeficiency syndrome (AIDS) patients but also cause of
progressive pulmonary disease even in immunocompe-
tent humans31. Clinical management of patients with M.
avium infections is difficult, even with macrolides such
as clarithromycin and azithromycin as first-line drugs
in multidrug regimens32. e prevalence of multi-drug
resistant and extensively drug resistant tuberculosis
(MDR- and XDR-TB) strains is reported to be high in the
countries where adequate supplies of the drugs are not
available. e frightening TB is due to use and misuse of
existing antibiotics and poor compliance with the long
duration of current chemotherapy. e current trends
suggest that TB will be among the 10 leading causes of
global disease burden in the year 202033. us, there is
an urgent need for development of new antitubercular
agents with improved properties such as enhanced activ-
ity against MDR and XDR strains, effectiveness against
latent TB, shortened duration of therapy, reduced toxic-
ity and rapid mycobactericidal mechanism of action34.
Although pharmaceutical industries have produced
a number of new antibiotics in the last three decades,
resistance by microorganisms has increased. Especially,
widespread use of antibiotics has led many bacteria to
evolve resistance to multiple versions of drugs such as
methicillin-resistant Staphylococcus aureus (MRSA),
vancomycin-resistant Enterococcus (VRE), and XDR-TB.
Generally, bacteria have the genetic ability to transmit and
acquire resistance to drugs, which are utilized as thera-
peutic agents and the concern is mainly on the number of
patients in hospitals who have suppressed immunity, and
due to new bacterial strains, which are multi-resistant35.
In our laboratory, a large number of Mannich bases
of kojic acid (route A), chlorokojic acid (route B), and
allomaltol (route C) were prepared (Scheme 1) for
their various types of biological activities4,6–11. Most of
these compounds seemed to be promising candidates
for anticonvulsant, antimicrobial and antiviral agents.
Mannich bases of kojic acid and allomaltol which
contain lipophilic aryl portions were synthesized by
our research group in order to increase the penetra-
tion to the blood-brain barrier4,6–9. ese compounds
demonstrated significant anticonvulsant activities in
vivo against maximal electroshock (MES)- and subcu-
taneous pentylenetetrazole (scPTZ)-induced seizure
tests. Also, in our recent studies Mannich bases of chlo-
rokojic acid (route B) exhibited good antimicrobial and
antiviral activities10–11
.
erefore in present work we planned to synthesize
eighteen novel 6-chloromethyl-3-hydroxy-2-substituted
4H-pyran-4-one derivatives including piperazine ring
and evaluate for their anticonvulsant and antimicrobial
couplet with antitubercular activities (Scheme 2).
Adding a phenyl ring or equivalent hydrocarbon sub-
stitute, and a carbonyl or another electronegative group
adjacent to the phenyl ring were expected to increase
lipophilicity and biological effect as succeeded before.
Materials-methods
Chemistry
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 omas Hoover Capillary
Melting Point Apparatus (Philadelphia, PA, USA) and
were uncorrected. IR spectra were recorded on a Perkin
Elmer FT-IR-420 System, Spectrum BX spectrometer. 1H-
NMR and 13C-NMR spectra were obtained with a Varian
Mercury 400 MHz spectrophotometer in deutorochlo-
roform (CDCl3) and dimethylsulphoxyde (DMSO-d6).
Tetramethylsilane (TMS) was used as an internal stan-
dard (chemical shift in δ, ppm). Mass spectral analy-
sis was carried out with a Micromass ZQ LC-MS with
Masslynx Software Version 4.1 by using electrospray
ionization (ESI+) method. e elemental analyses were
performed with a Elementar Analysensysteme GmbH
varioMICRO CHNS at e Scientific & Technological
Research Council of Turkey-Ankara Testing and Analyses
Laboratory (TUBİTAK-ATAL). For the compounds 9
and 17 elementary analysis were performed on Ankara
University, Faculty of Pharmacy, Central Laboratory on
CHNS-932 (LECO). e 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 in pre-
vious studies8–11. Yield 76%, m.p.: 166-167 °C.
Preparation of Mannich bases of chlorokojic acid derivatives
(1–18)
e secondary amine (substituted piperazine deriva-
tives) and 37% formaline were dissolved in MeOH.
Chlorokojic acid was added to the solution and the mix-
ture was stirred vigorously for 15 to 25 min. e result-
ing precipitate was collected by filtration and washed
with cold MeOH. All crude products recrystallized from
appropriate solvent.
Scheme 1. Schematic representation of the synthetic route A, B
and C.
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel chlorokojic acid derivatives
3
Scheme 2. General synthesis of compounds 1-18.
6-(Chloromethyl)-3-hydroxy-2-[(4-phenylpiperazin-1-yl)
methyl]-4H-pyran-4-one (1) IR ν (cm−1): 1622 (C=O), 1455
(C=C), 1201 (C-O); 757 (C-Cl); 1H-NMR (DMSO-d6, 400MHz)
δ ppm: 2.62 (4H; t; J= 4.8; piperazine-H2’, H6’), 3.13 (4H; t;
J= 4.8; piperazine-H3’,H5’), 3.63 (2H; s; -CH2-), 4.67 (2H; s;
ClCH2-), 6.56 (1H; s; H5), 6.76 (1H; t; J= 7.2; Ar-H4’’), 6.91
(2H; d; J= 8.0; H2’’ and Ar-H6’’), 7.19 (2H; t; J= 7.8; H3’’ and
Ar-H5’’), 9.24 (1H; brs; -OH); 13C-NMR (DEPT) (CDCl3,
400 MHz) δppm: 173.60, 112.08, 41.02, 49.13, 52.95, 55.26,
161.69, 150.99, 145.81, 129.17, 116.25, 120.09, 144.16; 13C-
NMR (APT) (CDCl3, 400 MHz) δ ppm: −55.26, −52.95,
−49.12, −41.22; ESI-MS (m/z): 195 (100%), 335 (M++H),
337 (M++H+2), 357 (M++Na).
(CDCl3, 400MHz) δ ppm: 2.44 (3H; s; CH3CO-), 2.61 (4H; t;
J= 5.0; piperazine-H2’, H6’), 3.33 (4H; t; J= 4.8; piperazine-
H3’, H5’), 3.63 (2H; s; -CH2-), 4.66 (2H; s; ClCH2-), 6.56 (1H;
s; H5), 6.95 (2H; d; J= 9.6; Ar-H2’’,H6’’) 7.79 (2H; d; J= 9.2;
Ar-H3’’, H5’’); ESI-MS (m/z): 377 (M++H), 379 (M++H+2),
399 (M++Na, 100%).
6-(Chloromethyl)-3-hydroxy-2-{[4-(4-nitrophenyl)piper-
azin-1-yl]methyl}-4H-pyran-4-one (6) IR ν (cm−1): 1630
1
(C=O), 1458 (C=C), 1201 (C-O); 753 (C-Cl); H-NMR (CDCl3,
400MHz) δ ppm: 2.48 (4H; t; J= 1.8; piperazine-H2’, H6’),
2.57 (4H; t; J= 4.8; piperazine-H3’, H5’), 3.61 (2H; s; -CH2-),
4.65 (2H; s; ClCH2-), 6.54 (1H; s; H5), 7.01 (2H; d; J= 9.6;
Ar-H2’’, H6’’), 8.02 (2H; d; J= 9.2; Ar-H3’’, H5’’), 9.34 (1H;
brs; -OH); 13C-NMR (DMSO-d6, 400 MHz) δ ppm: 42.09,
46.97, 52.51, 53.87, 113.12, 113.33, 126.35, 137.56, 144.83,
148.03, 155.35, 161.93, 174.15; ESI-MS (m/z): 325 (100%),
380 (M++H), 382 (M++H+2), 402 (M++Na).
6-(Chloromethyl)-3-hydroxy-2-[(4-o-tolylpiperazin-1-yl)
methyl]-4H-pyran-4-one (2) IR ν (cm−1): 1622 (C=O), 1453
1
(C=C), 1196 (C-O); 761 (C-Cl); H-NMR (CDCl3, 400 MHz)
δ ppm: 2.22 (3H; s; -CH3), 2.65 (4H; brs; piperazine-H2’,
H6’), 2.84 (4H; t; J= 4.4; piperazine-H3’, H5’), 3.65 (2H; s;
-CH2-), 4.67 (2H; s; ClCH2-), 6.57 (1H; s; H5), 6.9-7.10
(4H; m; Ar-H); 13C-NMR (DMSO-d6, 400 MHz) δ ppm:
17.43, 41.35, 51.19; 52.75; 53.39; 112.37; 118.64; 122.71;
126.40, 130.68, 131.62, 144.02, 147.59, 151.09, 161.13,
173.37; ESI-MS (m/z): 349 (M++H), 351 (M++H+2), 371
(100%, M++Na).
6-(Chloromethyl)-2-[(4-cyclohexylpiperazin-1-yl)methyl]-3-
hydroxy-4H-pyran-4-one (7) IR ν (cm−1): 1630 (C=O), 1450
1
(C=C), 1197 (C-O); 740 (C-Cl); H-NMR (CDCl3, 400MHz) δ
ppm: 1.06–1.71 (5H; m; cyclohexane-H); 1.54–1.71 (5H;
m; cyclohexane-H); 2.17 (1H; m; cyclohexane-H); 2.46
(8H; brs; piperazine-H), 3.53 (2H; s; -CH2-), 4.65 (2H;
s; ClCH2-), 6.53 (1H; s; H5); ESI-MS (m/z): 341 (M++H,
100%), 343 (M++H+2), 363 (M++Na).
6-(Chloromethyl)-3-hydroxy-2-[(4-p-tolylpiperazin-1-yl)
methyl]-4H-pyran-4-one (3) IR ν (cm−1): 1634 (C=O), 1456
1
(C=C), 1196 (C-O); 749 (C-Cl); H-NMR (CDCl3, 400MHz) δ
2-[(4-Benzylpiperazin-1-yl)methyl]-6-(chloromethyl)-3-
hydroxy-4H-pyran-4-one (8) IR (cm−1): 1627 (C=O), 1452
(C=C), 1200 (C-O); 748 (C-Cl); 1H-NMR (CDCl3, 400 MHz) δ
ppm: 2.37 (4H; brs; piperazine-H2’, H6’), 2.49 (4H; brs;
piperazine-H3’, H5’), 3.44 (2H; s; -CH2-), 3.55 (2H; s;
-CH2-Ar), 4.65 (2H; s; ClCH2-), 6.54 (1H; s; H5), 7.2–7.3
(5H; m; Ar-H); ESI-MS (m/z): 349 (M++H, 100%), 351
(M++H+2), 371 (M++Na).
ppm: 2.19 (3H; s; -CH3), 2.61 (4H; t; J= 4.8; piperazine-H2’,
H6’), 3.06 (4H; t; J= 5.0; piperazine-H3’, H5’), 3.62 (2H; s;
-CH2-), 4.67 (2H; s; ClCH2-), 6.56 (1H; s; H5), 6.81 (2H; d; J=
8.4; Ar-H2’’, Ar-H6’’) 7.01 (2H; d; J= 8.8; Ar-H3’’, Ar-H5’’), 9.24
(1H; brs; -OH); 13C-NMR (DMSO-d6, 400 MHz) δ ppm:
19.93, 41.33, 48.57, 52.24, 53.32, 112.34, 115.58, 127.53,
129.22, 144.02, 147.46, 148.79, 161.14, 173.37; ESI-MS
(m/z): 349 (M++H), 351 (M++H+2), 371 (M++Na, 100%).
6-(Chloromethyl)-3-hydroxy-2-{[4-(2-methylbenzyl)
6-(Chloromethyl)-2-{[4-(2,3-dimethylphenyl)piperazin-1-yl]
piperazin-1-yl]methyl}-4H-pyran-4-one (9) IR ν
(cm−1):
1
methyl}-3-hydroxy-4H-pyran-4-one (4) IR ν (cm−1): 1621
1622 (C=O), 1455 (C=C), 1197 (C-O); 746 (C-Cl); H-NMR
(CDCl3, 400MHz) δ ppm: 2.29 (3H; s; -CH3); 2.38 (4H; brs;
piperazine-H2’, H6’), 2.47 (4H; t; J= 1.8; piperazine-H3’,
H5’), 3.40 (2H; s; -CH2-), 3.55 (2H; s; -CH2-Ar), 4.65 (2H;
s; ClCH2-), 6.54 (1H; s; H5), 7.10–7.20 (4H; m; Ar-H); 13C-
NMR (DEPT) (CDCl3, 400 MHz) δ ppm: 173.85, 112.74,
41.43, 53.05, 53.39, 56.12, 161.45, 146.01, 137.75, 130.51,
125.72, 136.32, 130.02, 127.35, 144.55, 60.80, 19.46; 13C-
NMR (APT) (CDCl3, 400 MHz) δ ppm: −60.80, −56.12,
−53.39, −53.05, −41.43; ESI-MS (m/z): 363 (M++H), 365
(M++H+2), 385 (M++Na, 100%).
1
(C=O), 1454 (C=C), 1197 (C-O); 747 (C-Cl); H-NMR (CDCl3,
400MHz) δ ppm: 2.13 (3H; s; -CH3), 2.19 (3H; s; -CH3),
2.64 (4H; brs; piperazine-H2’, H6’), 2.79 (4H; t; J= 4.4; pip-
erazine-H3’, H5’), 3.64 (2H; s; -CH2-), 4.68 (2H; s; ClCH2-),
6.56 (1H; s; H5), 6.8–7.0 (3H; m; Ar-H); ESI-MS (m/z): 363
(M++H), 365 (M++H+2), 385 (M++Na, %100).
2 - { [ 4 - ( 4 - Ace t y l p h e n y l ) p i p e ra z i n - 1 - y l ] m e t h y l } - 6 -
(chloromethyl)-3-hydroxy-4H-pyran-4-one (5) IR ν (cm−1):
1
1622 (C=O), 1454 (C=C), 1199 (C-O); 747 (C-Cl); H-NMR
© 2012 Informa UK, Ltd.

4
G. Karakaya et al.
6-(Chloromethyl)-3-hydroxy-2-{[4-(3-methylbenzyl)piper-
6-(Chloromethyl)-2-{[4-(2,4-dichlorobenzyl)piperazin-1-yl]
azin-1-yl]methyl}-4H-pyran-4-one (10) IR ν (cm−1): 1624
methyl}-3-hydroxy-4H-pyran-4-one (16) IR ν (cm−1): 1626
1
1
(C=O), 1456 (C=C), 1197 (C-O); 747 (C-Cl); H-NMR (CDCl3,
(C=O), 1454 (C=C), 1199 (C-O); 738 (C-Cl); H-NMR (CDCl3,
400MHz) δ ppm: 2.28 (3H; s; -CH3); 2.36-2.50 (8H; m;
piperazine-H), 3.40 (2H; s; -CH2-), 3.56 (2H; s; -CH2-Ar),
4.63 (2H; s; ClCH2-), 6.52 (1H; s; H5), 7.03–7.19 (4H; m;
Ar-H); ESI-MS (m/z): 363 (M++H, 100%), 365 (M++H+2),
385 (M++Na).
400MHz) δ ppm: 2.50 (8H; brs; piperazine-H), 3.53 (2H;
s; -CH2-), 3.57 (2H; s; -CH2-Ar), 4.65 (2H; s; ClCH2-),
6.54 (1H; s; H5), 7.38-7.55 (3H; m; Ar-H), 9.20-9.40 (1H;
brs; -OH); 13C-NMR (DMSO-d6, 400 MHz) δ ppm: 42.09,
52.10, 54.08, 58.52, 113.11, 127.87, 129.31, 132.69, 134.74,
135.51, 144.74, 148.30, 161.85, 174.12; ESI-MS (m/z): 363
(M++H), 365 (M++H+2), 385 (M++Na, 100%).
6-(Chloromethyl)-3-hydroxy-2-({4-[3-(trifluoromethyl)
benzyl]piperazin-1-yl}methyl)-4H-pyran-4-one
(11) IR
ν (cm−1): 1623 (C=O), 1455 (C=C), 1197 (C-O); 749 (C-Cl);
1H-NMR (CDCl3, 400 MHz) δ ppm: 2.51 (8H; brs; pipera-
zine-H), 3.56 (2H; s; -CH2-), 3.57 (2H; s; -CH2-Ar), 4.65
(2H; s; ClCH2-), 6.54 (1H; s; H5), 7.53–7.61 (4H; m; Ar-H);
ESI-MS (m/z): 417 (M++H, 100%), 419 (M++H+2), 439
(M++Na).
6-(Chloromethyl)-2-{[4-(cyclohexylmethyl)piperazin-1-yl]
methyl}-3-hydroxy-4H-pyran-4-one (17) IR ν (cm−1): 1622
1
(C=O), 1456 (C=C), 1200 (C-O); 742 (C-Cl); H-NMR (CDCl3,
400MHz) δ ppm: 0.78–1.70 (11H; m; cyclohexane-H),
2.04 (2H; d; J= 7.6; -CH2-N=), 2.31 (4H; brs; piperazine-H2’,
H6’), 2.46 (4H; brs; piperazine-H3’, H5’), 3.54 (2H; s; -CH2-),
4.65 (2H; s; ClCH2-), 6.53 (1H; s; H5); ESI-MS (m/z): 355
(M++H, 100%), 357 (M++H+2), 377 (M++Na).
6-(Chloromethyl)-3-hydroxy-2-({4-[4-(trifluoromethyl)ben-
zyl]piperazin-1-yl}methyl)-4H-pyran-4-one (12) IR ν (cm−1):
1
6-(Chloromethyl)-2-{[4-(cyclohexanecarbonyl)piperazin-1
1623 (C=O), 1457 (C=C), 1198 (C-O); 749 (C-Cl); H-NMR
-yl]methyl}-3-hydroxy-4H-pyran-4-one (18) IR ν (cm−1):
(CDCl3, 400MHz) δ ppm: 2.40 (4H; brs; piperazine-H2’, H6’),
2.50 (4H; t; J= 8.8; piperazine-H3’, H5’), 3.55 (2H; s; -CH2-),
3.57 (2H; s; -CH2-Ar), 4.66 (2H; s; ClCH2-), 6.55 (1H; s; H5),
7.51 (2H; d; J= 7.6; Ar-H2’’, H6’’) 7.66 (2H; d; J= 8.0; Ar-H3’’,
H5’’); ESI-MS (m/z): 417 (M++H, 100%), 419 (M++H+2),
439 (M++Na).
1
1638 (C=O), 1447 (C=C), 1216 (C-O); 738 (C-Cl); H-NMR
(CDCl3,400 MHz)δppm:1.28–1.70(8H;m;cyclohexane-H),
2.40–2.46 (3H; m; cyclohexane-H), 2.51 (4H; brs; pipera-
zine-H2’, H6’), 3.45 (4H; brs; piperazine-H3’, H5’), 3.60 (2H;
s; -CH2-), 4.65 (2H; s; ClCH2-), 6.55 (1H; s; H5), 9.21 (1H;
brs; -OH); 13C-NMR (DMSO-d6, 400 MHz) δ ppm: 25.80,
26.25, 29.80, 41.59, 42.08, 45.42, 52.83, 53.50, 53.89,
113.11, 144.79, 148.08, 161.92, 174.03, 174.14; ESI-MS
(m/z): 325 (100%), 326, 369 (M++H), 371 (M++H+2),
391 (M++Na).
6-(Chloromethyl)-2-{[4-(2,5-difluorobenzyl)piperazin-1-yl]
methyl}-3-hydroxy-4H-pyran-4-one (13) IR ν (cm−1): 1627
1
(C=O), 1460 (C=C), 1052 (C-O); 732 (C-Cl); H-NMR (CDCl3,
400MHz) δ ppm: 2.38 (4H; brs; piperazine-H2’, H6’), 2.47
(4H; t; J= 1.8; piperazine-H3’, H5’), 3.40 (2H; s; -CH2-), 3.55
(2H; s; -CH2-Ar), 4.65 (2H; s; ClCH2-), 6.54 (1H; s; H5),
7.10–7.20 (3H; m; Ar-H); ESI-MS (m/z): 363 (M++H), 365
(M++H+2), 385 (M++Na, 100%).
Anticonvulsant activity
e compounds were tested for their anticonvulsant
activity against maximal electroshock (MES)- and sub-
cutaneous pentylenetetrazol (scPTZ)-induced seizure
threshold tests. e acute neurological toxicity was
determined in the rotorod test. All these tests were per-
formed in male mice according to the phase-I tests of the
Antiepileptic Drug Development (ADD) program which
were developed by National Institutes of Health (NIH),
National Institute of Neurological Disorders and Stroke
(NINDS). Stimulator (Grass S88, Astro-Med. Inc. Grass
Instrument Division, W. Warwick, RI, USA), constant
current unit (Grass CCU1A, Grass Medical Instrument,
Quincy, Mass., USA), and corneal electrodes were used
for the evaluation of anticonvulsant activity against
MES-induced seizure test. All synthesized compounds
were suspended in 30% aqueous of PEG 400 and
administered to the mice intraperitoneally in a volume
of 0.01 mL/g at body weight. Twelve Swiss albino male
mice (20 2 g) were used for each compound. Mice
were purchased from the Hacettepe University Animal
Farm according to the ADD-NINDS program36. All the
animals were acclimatized for a week before use. e
animals were maintained in colony cages under a 12
h-light-and-12 h-dark cycle and kept under standard
2 - { [ 4 - ( 4 - C h l o ro b e n z y l ) p i p e ra z i n - 1 - y l ] m e t hy l } - 6 -
(chloromethyl)-3-hydroxy-4H-pyran-4-one (14) IR ν (cm−1):
1
1621 (C=O), 1453 (C=C), 1196 (C-O); 746 (C-Cl); H-NMR
(CDCl3, 400MHz) δ ppm: 2.37 (4H; brs; piperazine-H2’,
H6’), 2.50 (4H; brs; piperazine-H3’, H5’), 3.44 (2H; s; -CH2-),
3.56 (2H; s; -CH2-Ar), 4.65 (2H; s; ClCH2-), 6.54 (1H; s; H5),
7.29 (2H; d; J= 8.8; Ar-H2’’, H6’’); 7.35 (2H; d; J= 8.4; Ar-H3’’,
H5’’), 9.18 (1H; brs; -OH); 13C-NMR (DMSO-d6, 400 MHz)
δ ppm: 41.31, 52.26, 53.32, 60.87, 112.32, 128.00, 130.42,
131.27, 137.17, 143.93, 147.56, 161.06, 173.33; ESI-MS
(m/z): 383 (M++H, 100%), 385 (M++H+2, % 66.07), 405
(M++Na).
6-(Chloromethyl)-2-{[4-(2,6-dichlorobenzyl)piperazin-1-yl]
methyl}-3-hydroxy-4H-pyran-4-one (15) IR ν (cm−1): 1620
1
(C=O), 1454 (C=C), 1196 (C-O); 766 (C-Cl); H-NMR (CDCl3,
400MHz) δ ppm: 2.49 (8H; brs; piperazine-H), 3.53 (2H;
s; -CH2-), 3.54 (2H; s; -CH2-Ar), 4.64 (2H; s; ClCH2-), 6.53
(1H; s; H5), 7.32 (1H; t; J= 8.0; Ar-H4’’), 7.44 (2H; d; J= 7.6;
Ar-H3’,’ H5’’); ESI-MS (m/z): 325 (100%), 419 (M++H,), 421
(M++H+2), 441 (M++Na).
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel chlorokojic acid derivatives
5
(hygienic) conditions at an ambient temperature of
22 3°C and at a relative humidity between 50 to 60%
and fed on standard laboratory diet and food and water
was provided ad libitum except at the time they were
brought out of the cage. All the experimental protocols
were carried out with the permission from Hacettepe
University, ‘Laboratory Animals Ethic Committee’ deci-
sion (02. 01. 2009 date 2008/ 80-4 number). Control ani-
mals received 30% aqueous PEG 400. Pentyleneterazol
was administered subcutaneously (s.c.) on the back of
the neck. e rotorod toxicity test was performed on a
1 inch diameter knurled wooden rod, rotating at 6 rpm
(the rotorod used in phase-I test was made by Hacettepe
University Technical Department). MES tests were elic-
ited with a 60-cycle alternating current of 50 mA inten-
sity (5–7 times more than that required to elicit minimal
seizures) delivered for 0.2 sec via corneal electrodes. A
drop of 0.9% saline was instilled into the eye prior to
application of the electrodes in order to prevent the
death of the animal. Abolition of the hind limb tonic
extension component of the seizure was defined as
protection. 85 mg/kg of pentyleneterazol (produces sei-
zures in more than 95% of mice) was administered as a
0.5% solution s.c. into the posterior midline. e animal
was observed for 30 min to decide whether the failure of
the threshold seizure (a single episode of clonic spasms
of at least 5 sec duration) could be defined as protec-
tion. e rotorod test was used to evaluate neurotoxic-
ity. e animal was placed on a 1 inch diameter knurled
wooden rod rotating at 6 rpm. Normal mice remain on a
rod rotating at this speed indefinitely. Neurologic toxic-
ity was defined as the failure of the animal to remain on
the rod for 1 min.
dispensed in each well of a sterile flat-bottom 96-well
plate, and serial twofold dilutions (256–0.06 µg/mL) of
each compound were prepared directly in the plate. One
hundred microliters of inoculum was added to each well. A
growth control and a sterile control were also included for
each stain. e plate was covered, and incubated at 37°C
under a normal atmosphere. After 7 days of incubation, 10
µg/mL of resazurin solution was added to each well, and
the plate was reincubated overnight. A change in colour
from blue (oxidized state) to pink (reduced) indicated the
growth of bacteria, and the minimum inhibitory concen-
tration (MIC) was defined as the lowest concentration of
drug that prevented this change in color.
Antibacterial and antifungal activities
e compounds of 1–18 were dissolved in
dimethylsulphoxide:ethanol (80:20) and sterilized by
filtration using 0.22 µm Millipore (MA 01730, USA) and
used as the stock solutions. Reference antibacterial
agents were purchased from Sigma Chemical Co. (St.
Louis, MO, USA) and dissolved in phosphate buffer solu-
tion (ampicillin, pH 8.0; 0.1 mol/mL), dimethylsulphox-
ide (ketoconazole), or in water (gentamicin, levofloxacin,
fluconazole)10,11. Antibacterial activity test were carried
out against standards; Gram negative standard strains of
Escherichia coli ATCC 35218, Pseudomonas aeruginosa
ATCC 10145, Proteus mirabilis ATCC 7002, Klebsiella
pneumoniae RSKK 574, Acinetobacter baumannii
RSKK 02026, and as Gram positive standard strains of
Staphylococcus aureus ATCC 25923, Enterococcus faecalis
ATCC 29212, Bacillus subtilis ATCC 6633 and their drug
resistant isolates were used for the determination of
antibacterial activity. Candida albicans ATCC 10231, C.
parapsilosis ATCC 22019, C. tropicalis ATCC 13803 and
C. krusei ATCC 6258 were used for the determination of
antifungal activity. Mueller-Hinton Broth (MHB; Difco)
and Mueller-Hinton Agar (MHA; Oxoid) were applied
for growing and diluting of the bacteria suspensions39.
e synthetic medium RPMI-1640 with L-glutamine was
buffered to pH 7 with 3-[N-morpholino]-propansulfonic
acid and culture suspensions were prepared as described
previously40. e broth microdilution method was
employed for antibacterial and antifungal activity tests.
Media were placed into each 96 wells of the microplates.
Extract solutions at 512 μg/mL were added into first rows
of microplates and twofold dilutions of the compounds
(256–0.125 μg/mL) were made by dispensing the solu-
tions to the remaining wells. e lowest concentration
of the compounds that completely inhibit macroscopic
growth was determined and MICs were reported as
Antitubercular activity
e strains Mycobacterium tuberculosis H37Rv (ATCC
27294; American Type Culture Collection) reference
strain and M. avium (ATCC 15769) were maintained
on Lowenstein–Jensen medium and subcultured on
Middlebrook 7H11 agar (Becton Dickinson) resuspended
in 7H9-S broth medium supplemented with 10% [OADC;
0.1% casitone, 0.5% glycerol, supplemented oleic acid,
albumin, dextrose, and catalase], 0.2% glycerol and 0.1%
Bacto casitone (Difco). Suspensions were prepared in
0.04% (vol/vol) Tween 80–0.2%+bovine serum albumin
so that adjusted to McFarland tube number 1. is was
diluted to 1:20 and 100 μl aliquot was used as inoculum.
Reference antibacterial agents of were purchased from
Sigma Chemical Co. (St. Louis, MO, USA) and dissolved
in dimethylsulphoxide (streptomycin), or in d-water
(isoniazid, ethambutol). e stock solutions of the agents
were prepared in medium according to the Clinical and
Laboratory Standards Institute (CLSI M38-A2, formerly
NCCLS)37,38. A stock solution of the resazurin sodium salt
(Sigma) powder was prepared at 0.01% in sterile distilled
water. It was filters-sterilized and kept at 4°C. One hun-
dred microliters of Middlebrook 7H9 broth (0.1% casitone,
0.5% glycerol, and 10% OADC; Becton-Dickinson) was
described previously study37,38,40
.
Results and discussion
Chemistry
Treatment of kojic acid with thionyl chloride at room
temperature yielded chlorokojic acid which after reduc-
tion with zinc and hydrochloric acid yielded allomaltol
© 2012 Informa UK, Ltd.

6
G. Karakaya et al.
(Scheme 1). ese three compounds provided basis for
our research area. Mannich bases of several hydroxy-
pyranones including kojic acid and pyromeconic acid
(3-hydroxy-4H-pyran-4-one) were synthesized before
by different researchers41,42. ey react with amines and
formaline like phenols to produce the Mannich base as a
result of aminoalkylation of the ortho position of the −OH
group.
logP (clogP) and elemental analysis data are presented
in Table 1. clogP refers to calculated hydrophobicity of
the compounds, respectively, clogP have been calcu-
lated from ACD/ChemSketch, Product version 12.01.
e thumb rules for clogP values, to a drug like molecule
must be lower than “5” to by-pass the cell barrier.
e structure of the compounds were clarified by IR,
1H-NMR and mass spectroscopy in experimental part.
Also, with a view to analyse the structures of compounds
1, 6, 9, 14, 16 and 18 13C-NMR spectroscopy was used.
e selected diagnostic bands of IR spectra of chlorokojic
acid derivatives provide useful information for determin-
ing structures. All the compounds exhibited absorbtion
bands about 1620cm−1 due to ν (C=O) streching of pyra-
none ring. Because of hydroxymethyl moiety showing two
hydrogen bondings both intra- and intermolecular, (C=O)
streching gave signals at lower frequency. IR spectra of all
compounds showed ν (C−Cl);at 732-766 cm−1, ν (C−O)
at 1200-1195cm−1 and ν (C=C) at 1447-1460cm−1. e
formation of the Mannich bases of chlorokojic acid was
further confirmed with the 1H-NMR spectra. Assignments
of the signals were based on the chemical shifts and
intensity pattern. e 1H-NMR spectra of the compounds
exhibited triplet peaks for -CH2- at piperazine between 2.2
and 3.6ppm. J values of these peaks were from 4.4 to 5.0
Hz. Phenyl ring’s protons exhibite signals at 7.6 to 9.2ppm.
Compounds 7, 17and 18which had cyclic protons in their
structure instead of phenyl ring, showed peaks ranging
0.78 to 2.17ppm as multiplets. Characteristic H5 proton
of the 4H-pyran-4-one ring were determined as singlet
peaks between 6.52 and 6.57ppm. Also, due to keto-enol
tautomerisation of hydroxyl group on pyranone ring −OH
peaks of compounds 2, 4, 5, 7, 9, 10, 11, 12 and 13 were
not observed. 13C NMR spectra analysis were supported
by DEPT and APT spectra which differentiate between
−CH=, -CH2- and -CH3 groups. e 13C NMR signals of
compounds 1, 6, 9, 14, 16 and 18 were in good agree-
ment with proposed structures. Compounds displayed
characteristic peaks of methylene group (ClCH2-) carbons
at 41.02, 42.09, 41.43 41.31, 42.09 and 42.08ppm, respec-
tively. Carbonyl carbons of the 4H-pyran-4-one ring was
found at a range of 161.06-161.93ppm. Signals at 150.99-
146.01ppm were due to C3 of the pyranone ring whereas
112.08-113.12ppm belong to C5. e distinctive signals of
all compounds were observed in the mass spectra which
followed the similar fragmentation pattern. e entire
spectrums showed molecular ion peaks, M++23 (Na)
peaks and isotope peaks owing to chlorine atom.
In an earlier study, it was reported that di-Mannich
derivatives were obtained in an acidic medium from the
reaction of kojic acid, formaline and aromatic amine43.
On the other hand, although there were two open nuclear
positions of kojic acid (3- and 6-), because of its phenol-
like properties, the reaction occurred only at 6-position
and mono-Mannich derivatives were obtained in basic
medium (Scheme 3). When the enolic hydroxyl group
converted to an ether group, 6-position was deacti-
vated41. e mechanisms of this Mannich reaction both
in an acidic and a basic medium were investigated and
found that, enhanced reactivity in a basic medium is due
to increase of the electronegativity at 6-position42.
It is well known that chlorokojic acid is an important
compound from the chemical point of view and as the
chlorine atom in the structure readily undergoes nucleo-
philic substitution44, it has been used as starting or inter-
mediate material in many reactions2–4,6–11
.
General synthesis of compounds is given in Scheme 2.
Chlorokojic acid was gained by commercially available
kojic acid as described methods from the published
literature8–11. In order to obtain chlorokojic acid; 2-hy-
droxymethyl moiety of kojic acid was chlorinated by
using thionyl chloride at room temperature.
Mannich type reactions are three component con-
densation reactions involving carbonyl compounds
which exist as keto-enol tautomeric forms, formaline
and a secondary amine. e amino alkylation of aro-
matic substrates by Mannich reaction is of considerable
importance for the design, synthesis and modification of
biologically active molecules. It has advantages ranging
from lower reaction times, increased reaction rates to
higher yields and reproducibility45. In order to investi-
gate the influence of secondary amines moieties such as
piperidine, morpholine and piperazine in the structure
of Mannich bases, all of them are used in our previous
studies (Scheme 2)4,6–11. Herein the basic substituent was
introduced at the 6-position of chlorokojic acid via a
Mannich type reaction using formaline and an appropri-
ate substituted piperazine.
e physicochemical parameters of the synthesized
compounds including melting point, yield, calculated
Anticonvulsant activity
Seizures that are arising from discharging lesions of the
cerebral cortex often occur as part of an epileptic syn-
drome. It is a group of signs and symptoms that custom-
arily occur together. Identification of the syndrome helps
to determine the appropriate therapy and the prognosis.
e MES-induced seizure test is a predictor of com-
pounds that are active against tonic-clonic (grand mal)
seizures. e scPTZ-induced seizure test is used to detect
Scheme 3. Numeration and tautomers of kojic acid in basic
medium.
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel chlorokojic acid derivatives
7
Table 1. Physicochemical parameters of the synthesized compounds.
Com
no
Mol. Formula
(Mol. Wt.)
M. p.
(°C)
Log ε
Yield
Elemental analysis
R
cLogP*
(λmax)nm (%)
Found % (calculated)
C
H
N
1
phenyl
C17H19ClN2O3
(334.80)
155-6 2.67 0.75
151-2 3.14 0.75
168-9 3.14 0.75
164-5 3.60 0.75
163-4 2.45 0.75
178-9 3.12 0.75
164-5 2.35 0.75
150-1 1.82 0.75
152-3 2.28 0.75
147-9 2.28 0.75
161-2 2.40 0.75
150-1 2.40 0.75
151-2 1.99 0.75
168-9 2.42 0.75
167-9 3.03 0.75
151-2 3.03 0.75
151-2 2.88 0.75
165-7 2.58 0.75
4.37
(205)
4.44
88
70
67
88
85
66
75
63
91
89
78
83
75
92
90
85
76
75
60.28
(60.99)
61.83
5.51
8.32
(8.37)
7.92
(5.72)
5.89
2
2-methylphenyl
4-methylphenyl
2,3-dimethylphenyl
4-acetylphenyl
4-nitrophenyl
cyclohexyl
C18H21ClN2O3
(348.82)
(207)
4.52
(61.98)
61.72
(6.07)
5.82
(8.03)
7.84
3
C18H21ClN2O3
(348.82)
(203)
4.42
(61.98)
62.54
(6.07)
6.13
(8.03)
7.74
4
C19H23ClN2O3
(362.85)
(211)
4.34
(62.89)
59.46
(6.39)
5.40
(7.72)
7.43
5
C19H21ClN2O4
(376.83)
(202)
4.32
(60.56)
53.95
(5.62)
4.68
(7.43)
10.88
6
C17H18ClN3O5
(379.80)
(202)
4.17
(53.76)
59.71
(4.78) (11.06)
7
C17H25ClN2O3
(340.85)
7.19
(7.39)
6.19
8.08
(8.22)
7.46
(222)
4.33
(59.90)
61.44
8
benzyl
C18H21ClN2O3
(348.82)
(206)
4.27
(61.98)
62.52
(6.07)
6.57
(8.03)
7.76
9
2-methylbenzyl
3-methylbenzyl
3-trifluoromethylbenzyl
4-trifluoromethylbenzyl
2,5-difluorobenzyl
4-chlorobenzyl
2,6-dichlorobenzyl
C19H23ClN2O3
(362.85)
(202)
4.27
(62.89)
63.16
(6.39)
6.08
(7.72)
7.74
10
11
12
13
14
15
16
17
18
C19H23ClN2O3
(362.85)
(203)
4.24
(62.89)
54.69
(6.39)
4.45
(7.72)
6.73
C19H20ClF3N2O3
(416.82)
(211)
3.97
(54.75)
54.59
(4.84)
4.75
(6.72)
6.71
C19H20ClF3N2O3
(416.82)
(216)
3.99
(54.75)
55.88
(4.84)
4.91
(6.72)
7.28
C18H19ClF2N2O3
(384.81)
(210)
4.35
(56.18)
56.08
(4.98)
5.10
(7.28)
7.31
C18H20Cl2N2O3
(382.90)
(221)
4.59
(56.41)
51.94
(5.26)
4.62
(7.31)
6.57
C18H19Cl3N2O3
(417.71)
(203)
4.42
(51.76)
51.57
(4.58)
4.58
(6.71)
6.66
2,4-dichlorobenzyl
cyclohexylmethyl
cyclohexylcarbonyl
C18H19Cl3N2O3
(417.71)
(204)
4.17
(51.76)
60.89
(4.58)
7.41
(6.71)
7.98
C18H27ClN2O3
(354.87)
(201)
4.32
(60.92)
57.87
(7.67)
6.65
(7.89)
7.33
C18H25ClN2O4
(368.85)
(202)
(58.61)
(6.83)
(7.59)
*cLogP: values are calculated theoryticaly by ACD/ChemSketch, Product version 12.01.
compounds useful in treating generalized absence (petit
mal) seizures36.
Anticonvulsant activity tests were performed in male
mice according to the phase-I tests of the ADD program
which were developed by NIH and NINDS36. Herein, the
anticonvulsant activities of the synthesized compounds
were evaluated by MES and scPTZ-induced seizure tests
performed at 0.5 and 4 h after administration with 30,
100 and 300 mg/kg doses using male Swiss albino mice
(20 2 g). e acute neurological toxicity was deter-
mined in the rotorod test. e results are presented in
Table 2.
According to our previous studies, when route A and C
(Scheme 1) were examined, it was seen that substituted
phenylpiperazine derivatives bearing 3-trifluoromethyl,
4-fluoro, 2-methoxy, 2-chloro and 4-chloro moieties
were the most protective compounds in the Mannich
series against convulsions at all doses. Also, kojic acid
and allomaltol derivatives that carry hydroxypiperi-
dine moiety were significantly more protective against
scPTZ and MES tests at all doses9 than other piperidine
Chlorokojic acid was not found protective against
scPTZ-induced seizure test; however, some series of
derivatives6,7
.
© 2012 Informa UK, Ltd.

8
G. Karakaya et al.
Table 2. Anticonvulsant and neurotoxicity screening data of the synthesized compounds.
MESa ScPTZb
0.5h (mg/kg)
30 100 300
Toxicityc
0.5h (mg/kg)
30 100 300
0.5h (mg/kg)
4h (mg/kg)
100 300
4h (mg/kg)
30 100 300
4h (mg/kg)
30 100 300
Compounds
30 100 300 30
1
2
3
4
5
6
7
8
0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 1/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 0/1 0/1 0/1 1/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 1/1 1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 1/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 1/1 1/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 1/1 0/1 0/1 1/1 1/1 1/1 1/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 1/1 0/1 1/1 1/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 1/1 1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
0/1 0/1 1/1 0/1 0/1 1/1 0/1 0/1 0/1 0/1 0/1 0/1 0/4 0/4 0/4 0/2 0/2 0/2
9
10
11
12
13
14
15
16
17
18
CKA
CKA: Chlorokojic Acid, aMES: Maximal electroshock, bScPTZ: Subcutaneous pentylenetetrazol, cToxicity: Rotorod test, 0/1: no activity,
1/1: noticeble activity.
chlorokojic acid achieved notable activity when their
Mannich bases were examined. Compounds 12 and 14
showedprotectionatboth300mg/kgand100mg/kgdoses
at 4h. It may be speculated that this is probably because
of increment in lipophilicity. Also, when halogen substi-
tution to benzyl ring is examined, at all doses, 2,5-diflu-
orobenzyl (compound 13) derivative was determined as
the most active compound by scPTZ-induced seizure test
at 4h, but in contrast when 3,4-dichlorobenzyl derivative
(compound 16) was tested instead of fluoro atom, scPTZ
protection was failed. However this time, selective MES
protection was observed at 300 and 100 mg/kg. None of
the compounds 4, 5, 7, 10, 15, 17 and 18 showed anticon-
vulsant activity.
With respect to the results of the MES tests, chlorokojic
acid showed protection at the 300mg/kg dose at both at
0.5 and 4h. e augmentation of this activity is aimed by
preparing Mannich bases. Moreover, compounds 9 and 16
bearing 2-methylbenzyl and 2,4-dichlorobenzyl moiety,
respectively, were found as the most effective molecules
with selective protection at 100 and 300mg/kg doses at 4h
in this series. Against the same test, compounds 1, 6and 13
exhibited activity at the 300mg/kg. At the same dose, com-
pounds 2, 3, 6, 8 and 12 also showed protection against
scPTZ-induced seizure test. None of the compounds
showed neurotoxicity at any of the studied doses.
protective. Finally, anticonvulsant activities of Mannich
bases were increased in some compounds while some
were decreased when compared to chlorokojic acid.
When the results of this study were compared to our
previous studies4,8,9 the expected increment in anticon-
vulsant activity of Mannich bases could not be observed.
at shows us chlorokojic acid derivatives of Mannich
bases have lower biological activities than kojic acid and
allomaltol derivatives.
Antitubercular activity
e antitubercular activity of the compounds was per-
formed as MICs against M. tuberculosis and M. avium
by using Resazurin microplate assay procedure (REMA).
Isoniazid, ethambutol and streptomycin were used as
reference compounds (Table 3).
Mannich bases of allomaltol derivatives including
3-methyl, 4-methyl and 3,5-dimethyl piperidine and
2-methoxyphenyl piperazine containing piperazine and
piperidine structure which were firstly synthesized by
our research group were found to have antimycobacterial
effect against Mycobacterium smegmatis46,47. However the
main cause of TB is M. tuberculosis. It divides extremely
slower than the other bacteria but can be cultured in
vitro whereas the others can only grow within the cells of
a host48. M. avium is a part of an nontuberculous myco-
bacteria group which causes pulmonery diseases resem-
bling TB. Herein, the in vitro antitubercular activity of the
compounds against M. tuberculosis and M. avium was
evaluated and MIC values are demonstrated in Table 3.
All of the Mannich bases have antitubercular activity and
have shown promising in vitro antitubercular activity
When the effect of aromaticity was examined, it was
seenthat,withthereductionofphenylincompound1and
benzyl group in compound 8 to change into cyclohexyl
(compound 7) and cyclohexylmethyl (compound 17),
respectively, their anticonvulsant activities disappeared.
Also, compound 18 bearing cyclohexylcarbonyl was not
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel chlorokojic acid derivatives
9
Table 3. Screening for antimicrobial activity against Gram positive bacteria and Mycobacterium (MIC in µg/mL).
Gram positive bacteria
Mycobacterium
S. aureus
E. faecalis
B. subtilis
M. tuberculosis M. avium
ATCC 25923 Isolated strain ATCC 29212 Isolated strain ATCC 6633 Isolated strain
ATCC 27294
ATCC 15769
1
2
3
4
5
6
7
8
16
16
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
>128
128
32
32
32
32
32
32
32
32
32
32
32
16
32
64
64
64
64
64
16
0.5
0.5
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
>128
32
16
16
8
32
32
32
32
32
32
32
32
32
32
32
16
32
64
64
64
64
64
64
0.5
-
32
32
8
16
16
8
16
16
8
16
16
32
16
32
32
32
16
16
32
16
16
16
32
16
16
16
16
16
8
16
8
16
8
16
8
16
8
32
32
32
8
9
16
8
10
11
12
13
14
15
16
17
18
CKA
AMP
LVX
INH
EMB
SM
16
8
16
8
8
4
8
16
8
8
32
16
16
16
32
16
8
8
32
8
32
8
64
8
32
4
32
8
<0.12
0.25
0.12
-
0.125
0.125
2
1
2
2
CKA: Chlorokojic acid, AMP: Ampicilline, LVX: Levofloxacin, INH: Isoniazid, EMB: Ethambutol, SM: Streptomycin
Isolated strain of S. aureus (methicillin resist; MRSA), isolated strain of E. faecalis (cephalosporin resist), isolated strain of B. subtilis
(ceftriaxon resist).
against M. tuberculosis in a MIC range of 8-32 µg/mL.
Among the entire series the most effective one was com-
pound 3 carrying 4-methylphenyl piperazine structure
against M. tuberculosis (MIC: 8 µg/mL). e antituber-
cular activity against M. avium was generally stronger
with MIC values of 4-32 µg/mL. Compound 18 bearing
carboxyphenyl piperazine moiety showed the highest
antitubercular activity (MIC: 4 µg/mL) against M. avium
and comparable results to reference drug ethambutol
and stretomycin (MIC: 2 µg/mL). Generally, compounds
showed greater inhibition activity over M. avium growth
than M. tuberculosis whereas compounds 3 and 8-10 had
the same MIC values against both microorganisms. e
existing antimycobacterial activity of chlorokojic acid
was improved by synthesizing their Mannich bases at
only compounds 3 and 18. However when compared to
reference drugs (isoniazid, ethambutol, streptomycin)
antimycobacterial activity were not strong as expected
and the scaffold should be developed in order to obtain
more active compounds.
Laboratory Standards Institute (CLSI)10,11. For antibacte-
rial activity assessment, standard strains (Escherichia coli,
Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella
pneumoniae, Acinetobacter baumannii, Staphylococcus
aureus, Enterococcus faecalis and Bacillus subtilis) and
their drug-resistant isolates were tested; and for anti-
fungal activity Candida albicans and C. parapsilosis, C.
tropicalis and C. krusei were used.
Ampicillin, gentamicin, levofloxacin for antibacte-
rial, ketoconazole and fluconazole for antifungal assays
were also tested under identical conditions. e results
are demonstrated in Tables 3–5 and expressed as MIC
values in comparison to reference drugs and chlorokojic
acid.
Some of the important antifungal agents (e.g. posacon-
azole,ketoconazole,itraconazole)thatarebeingusedforthe
treatment of fungal infections and antibiotics such as newly
marketed linezolid, contain a piperazine and/or an azole
ring in their structures11. Since the compounds we synthe-
sized previously have remarkable antimicrobial activities,
we aimed to assess the contribution of the substituents on
the piperazine structure to the biological activity.
Accordingtoourpreviousstudies10,11,6-(chloromethyl)-
3-hydroxy-2-[(3,4-dichlorobenzylpiperazin-1-yl)
methyl]-4H-pyran-4-one were significantly more active
Antibacterial and antifungal activity
e newly synthesized compounds (1–18) were tested
for their in vitro antibacterial and antifungal activi-
ties according to the guidelines of the Clinical and
© 2012 Informa UK, Ltd.

10 G. Karakaya et al.
than 3,4-dichlorophenyl derivative. However, in the
compound containing fluorine atom, no meaningful
changes were observed in activity (Figure 1). Our efforts
for the synthesis of Mannich bases possessing antimicro-
bial activity led us to find out that compounds bearing
chlorine atom were effective molecules. Also, to discover
the effect of methylene linkage in compounds, we syn-
thesized both the phenyl and the benzyl derivatives of
the same substituents10,11. In the light of these facts, we
examined the activities of designed Mannich bases bear-
ing benzyl piperazine.
e synthesized compounds showed a broad spec-
trum of antibacterial activity against Gram negative and
Gram positive standard strains with MIC values between
4 and 64 μg/mL (Table 3 and 4). In the meantime, the
compounds showed activity against drug-resistant iso-
lated both Gram positive and negative strains with MIC
values of 16 to 128 μg/mL.
As observed for Gram negative bacteria, compounds
1 and 11, bearing phenyl piperazine and 3-trifluorom-
ethylbenzyl moieties respectively, were the most active
compounds with the same MIC values (8–16 µg/mL).
Both of them were four-folds more active than chlorokojic
acid against E. coli. In this case, against the other Gram
negative bacteria, activity increased two-folds. Structural
modifications were not effective on activity, because the
other compounds (compounds 2–11; 12–18) had the
same MIC values with chlorokojic acid (16–32 µg/mL).
As seen in Table 4, compound 12 having 4-triflu-
oromethylbenzyl in its structure was determined to
have significantly high antibacterial potential against
standard strains of Staphylococcus aureus and Bacillus
subtilis with an inhibition between 4 and 8 µg/mL. In
comparison of compound 12 with chlorokojic acid
(MIC: 8-32 µg/mL) against both of mentioned bacteria,
its activity increased four and two times, respectively.
Moreover, compounds 1-12 and 13 also showed higher
activity with MIC value 16 µg/mL than chlorokojic acid
(MIC: 32 µg/mL) whereas compounds 14-16 and 18
had no difference in activity with chlorokojic acid. e
least efficiency of the compounds among Gram positive
bacteria was seen on Enterococcus facealis with a con-
centration of 32-64 µg/mL except compound 13 (MIC:
16 µg/mL). Among the series of compounds (14–16)
bearing chlorobenzylpiperazine the expected incre-
ment, like in 3,4-dichloro derivatives (Figure 1), was
not observed in antimicrobial activity. us, it could
be understood that within benzyl series the location of
chlorine substituents affects the activity. As references
ampicillin, gentamicin, and levofloxacin the tested com-
pounds bearing slight activity against tested standard
and their drug resistant isolates (E. coli, P. aeruginosa,.
Table 4. Screening for antimicrobial activity against Gram negative bacteria (MIC in µg/mL).
Gram negative bacteria
E. coli
Isolated
ATCC 35218 strain ATCC 10145 strain
P. aeruginosa
P. mirabilis
K. pneumoniae
Isolated
strain RSKK 02026
A. baumannii
Isolated
Isolated
Isolated
strain
ATCC 7002
strain
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
>128
1
RSKK 574
1
2
3
4
5
6
7
8
8
32
32
32
32
32
32
32
32
32
8
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
>128
0.5
16
32
32
32
32
32
32
32
32
32
16
32
32
32
32
32
32
32
32
-
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
-
8
16
16
16
16
16
16
16
16
16
8
8
16
16
16
16
16
16
16
16
16
8
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
>128
1
8
16
16
16
16
16
16
16
16
16
8
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
128
>128
64
9
10
11
12
13
14
15
16
17
18
CKA
AMP
LVX
GM
32
32
32
32
32
32
32
32
2
16
16
16
16
16
16
16
16
2
16
16
16
16
16
16
16
16
2
16
16
16
16
16
16
16
16
2
0.12
-
1
64
<0.12
-
0.12
-
0.12
-
-
0.5
2
-
-
-
CKA: Chlorokojic acid, AMP: Ampicilline, LVX: Levofloxacin, GM: Gentamicine.
E. coli isolates; (+ESβLs enzyme), P. aeruginosa isolates (resist to Trimethoprim-sulfamethoxazole, tazobactam), P. mirabilis isolates
isolates (resist to Trimethoprim-sulfamethoxazole, amoxicillin clavulonat, cefriaxon), K. pneumonae; isolates (resist to Trimethoprim-
sulfamethoxazole, amoxicillin clavulonat, cefriaxon), A. baumannii isolates (Trimethoprim-sulfamethoxazole resist).
Journal of Enzyme Inhibition and Medicinal Chemistry

Novel chlorokojic acid derivatives 11
P. mirabilis,.K. pneumoniae, A. baumannii, S. aureus,
E. faecalis, B. subtilis).
Conclusion
In present study, Mannich bases of chlorokojic acid were
synthesized and screened for their biological activities.
Compounds were designed in such a way that the pyra-
none nucleus was substituted with different piperazine
derivatives containing phenyl, benzyl or cyclohexyl
groups. e existing anticonvulsant activity of chlorokojic
acid, which was active against MES-induced seizure
test, was aimed to increase by preparing more lipophilic
agents as Mannich derivatives. e results of anticon-
vulsant evaluation revealed that compound 13, bearing
2,4-difluorobenzyl moiety showed the highest protection
against scPTZ-induced seizures. When antimicrobial
activity was evaluated, compounds 1 and 11, bearing
phenyl piperazine and 3-trifluoromethylbenzyl moieties
respectively, were found as the most active compounds
with MIC values of 8-16 µg/mL against Gram negative
bacteria.Compound12,carrying4-trifluoromethylbenzyl
group, was significantly effective among the synthesized
compounds and chlorokojic acid against Gram positive
bacteria. According to the antifungal activity results, the
existing activity of chlorokojic acid increased, especially
compound 1 was established as the most effective mol-
ecule against Candida spp. Beside this, compound 18
has been identified as a promising lead molecule for
antimycobacterial activity with MIC value that is com-
parable to reference drugs. e endpoint in evaluation
all of activity in piperazine containing Mannich bases of
chlorokojic acid in this study is, these compounds have
lower biological activities unexpectedly. However these
compounds would represent a productive matrix for the
development of new biologically important agents and
deserve further investigation.
According to the obtained data (Table 5), com-
pounds 1–3, having phenyl, 2- and 4-methylphenyl
in their structures respectively, possessed significant
antifungal activity against C. krusei with MIC value of
32 µg/mL, even more active than the reference drug,
fluconazole and chlorokojic acid, while compounds
4–18 had the same (MIC: 64 µg/mL). e MIC values
of compounds 1 and 12 were 8 µg/mL with showing the
highest activity against C. albicans in this series. Also,
compounds 3–11, 14 and 16 showed more remarkable
antifungal activity with MIC value of 16 µg/mL than
chlorokojic acid (MIC: 32 µg/mL). Compounds 1-3
inhibited the growth of C. tropicalis two-folds (MIC: 32
µg/mL) than chlorokojic acid and compounds 11, 15
and 16 (MIC: 64 µg/mL). Compared with reverences
all tested compounds shows weak antifungal activity
against Candida species (C. albicans, C. parapsilosis, C.
tropicalis) except from C. krusei with a MIC values of
fluconazole 64 µg/mL.
Figure 1. Phenyl and benzylpiperazine derivatives of Mannich
bases of chlorokojic acid.
Table 5. Screening for antifungal activity of the compounds (MIC
in µg/mL).
Fungus
C. albicans C. parapsilosis C. tropicalis
C. krusei
Acknowledgement
ATCC 10231 ATCC 90028 ATCC 13803 ATCC 6258
We would like to thank Professor Erhan Palaska for his
help with the mass spectra analysis. e Ankara University
Central Laboratory, Faculty of Pharmacy provided support
1
2
3
4
5
6
7
8
8
32
16
16
16
16
16
16
16
16
16
8
32
32
32
64
64
64
64
64
64
64
32
64
64
64
64
64
64
64
32
1
32
32
32
32
32
64
64
64
64
64
64
64
64
64
64
64
64
64
64
64
64
4
32
1
for acquisition of the H and 13C NMR spectrometer and
128
128
128
128
128
128
128
64
CHNS-932 (LECO) used in this work.
Declaration of Interest
is project was supported as a M.Sc. thesis by Hacettepe
University Scientific Research Fund (Project no.
09D01301002-4756). e authors state that there is no
conflict of interest.
9
10
11
12
13
14
15
16
17
18
CKA
KET
FLU
128
128
128
64
16
16
32
16
32
32
32
0.5
2
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