Mannich base derivatives of 3-hydroxy-6-methyl-4H-pyran-4-one with antimicrobial activity

Article abstract of DOI:10.3906/kim-0908-214

A series of 3-hydroxy-6-methyl-2-[(substitutedpiperidine-1-yl)methyl]-4H- pyran-4-one structured compounds were synthesized by reacting 5-hydroxy-2-methyl-4H-pyran-4-one with suitable piperidine derivatives using Mannich reaction conditions. Antibacterial activities of the compounds for E. coli ATCC 25922, S. paratyphi ATCC BAA-1250, S. flexneri ATCC 12022, E. gergoviae ATCC 33426, and M. smegmatis ATCC 14468 were assessed in vitro by the broth dilution method for determination of minimum inhibitory concentration (MIC). In addition, their inhibitory effects over DNA gyrase enzyme were evaluated using a DNA gyrase supercoiling assay. All the synthesized compounds showed a MIC value of either 8 or 16 μg/mL for M. smegmatis, whereas minimum to moderate activity was achieved for the others. Those tested in the supercoiling assay had at best a very mild inhibition of the enzyme. This series deserves further attention for testing over Mycobacterium species and topoisomerase II inhibition to develop new lead drugs to treat non-tuberculous mycobacterial infections. TUBITAK.

Full text of DOI:10.3906/kim-0908-214

Turk J Chem
4 (2010) , 447 – 456.
3
¨
˙
ꢀ
c TUBITAK
doi:10.3906/kim-0908-214
Mannich base derivatives of 3-hydroxy-6-
methyl-4H-pyran-4-one with antimicrobial activity
1
1
¨
1
2
¨
3
Demet US , Barkın BERK , Ece GURDAL , Nihan AYTEKIN , Tanıl KOCAGOZ ,
˘
2
2
˙
1,∗
Berrak C¸ AGLAYAN , I ¸s ıl AKSAN KURNAZ , Dilek DEMIR EROL
1
Yeditepe University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry,
˙
3
4755, Kayı ¸s da ˘g ı, Istanbul-TURKEY
e-mail: derol@yeditepe.edu.tr
2
Yeditepe University, Faculty of Engineering and Architecture, Department of Genetics and
˙
Bioengineering, 34755, Kayı ¸s da ˘g ı, Istanbul-TURKEY
3
Acıbadem University, Faculty of Medicine, Department of Microbiology,
˙
Istanbul-TURKEY
Received 15.10.2009
A series of 3-hydroxy-6-methyl-2-[(substitutedpiperidine-1-yl)methyl]-4H -pyran-4-one structured com-
pounds were synthesized by reacting 5-hydroxy-2-methyl-4H -pyran-4-one with suitable piperidine deriva-
tives using Mannich reaction conditions. Antibacterial activities of the compounds for E. coli ATCC 25922,
S. paratyphi ATCC BAA-1250, S. flexneri ATCC 12022, E. gergoviae ATCC 33426, and M. smegmatis
ATCC 14468 were assessed in vitro by the broth dilution method for determination of minimum inhibitory
concentration (MIC). In addition, their inhibitory effects over DNA gyrase enzyme were evaluated using a
DNA gyrase supercoiling assay. All the synthesized compounds showed a MIC value of either 8 or 16 μg/mL
for M. smegmatis, whereas minimum to moderate activity was achieved for the others. Those tested in the
supercoiling assay had at best a very mild inhibition of the enzyme. This series deserves further attention
for testing over Mycobacterium species and topoisomerase II inhibition to develop new lead drugs to treat
non-tuberculous mycobacterial infections.
Key Words: Antimycobacterial and antibacterial activity; DNA gyrase activity; Mannich bases of 3-
hydroxy-4H -pyran-4-one.
∗
Corresponding author
4
47

Mannich base derivatives of 3-hydroxy-6-methyl-4H-pyran-4-one..., D. US, et al.,
Introduction
1
The genus Mycobacterium has 140 well characterized species with its own family Mycobacteriaceae. This
genus includes saprophytic species that are widespread in nature, as well as causative pathogens of major
human diseases like M. tuberculosis, M. bovis, M. africanum, and M. microti, which cause tuberculosis, and M.
2
leprae, which causes leprosy. Mycobacteria referred to as atypical mycobacteria, non-tuberculous mycobacteria
(NTM), or mycobacteria other than tuberculous bacillus (MOTT) can produce localized disease in the lungs,
3
,4
lymph glands, skin, wounds, or bone, especially in susceptible/immunocompromised individuals.
Infections
5
and diseases due to non-tuberculous mycobacteria (NTM) are rising. There are no genome sequence data and
6
understanding of the biology of these ‘atypical mycobacteria’ is limited.
Mycobacteria do not contain endospores or capsules. They are neither gram-positive nor gram-negative
in the traditional sense; they are classified as acid-fast gram-positive bacteria due to a lack of outer cell
membrane. All Mycobacterium species share a characteristic cell wall, thicker than in many other bacteria,
7
which is hydrophobic, waxy, and rich in mycolic acids/mycolates. Transportation of compounds through the
cell wall of mycobacteria is one major factor relevant to the design of new antimycobacterial agents. This is
difficult since mycolic acids and surface associated lipids of these organisms form a transport barrier compared
8
to the cell wall of other eubacteria.
Quinolones (1), naphthyridones (2), and coumarins (3) inhibit bacterial type II topoisomerases, deoxyri-
bonucleic acid (DNA) gyrase, and topoisomerase IV, and are essential enzymes that maintain the supercoils in
9
,10
DNA both in gram-positive and gram-negative bacteria
(Figure 1).
Topoisomerase IV genes that have been reported in most bacterial species, are absent in a few bacteria
1
1
such as Treponema spp., Helicobacter spp., and notably Mycobacterium spp. and this is another factor that
limits the antimycobacterial design. Consequently, these bacteria are unusual in producing a unique type II
1
0
topoisomerase.
Quinolones (1), naphthyridones (2), and coumarins (3) are relatively functional in Mycobacterium
therapy with their low lipid solubility and rapidly increasing multi-drug resistance. Different Mannich bases
of hydroxypyranones by means of piperidine and piperazine side chains have structural resemblance with these
stated structures and might be an alternative for therapy.
Since Yabuta first synthesized allomaltol from kojic acid, which is a metabolic product of several species
1
2
of the genus Aspergillus,
many researchers have studied Mannich bases of kojic acid (4), maltol, and
1
3−17
18,19
20−22
allomaltol (6) derivatives to improve their antimicrobial,
antitumor,
anticonvulsant,
tyrosinase
2
3,24
12,15,16,20−22,25,26
inhibitory,
and toxic side effects.
These are generally based on their high iron chelating
2
7,28
capacities and high lipophilic characters
(Figure 1).
Mannich bases of hydroxypyranones have high lipid solubility and structural likeness to the above-
mentioned compounds that inhibit bacterial type II topoisomerases, DNA gyrase, and topoisomerase IV. We
have focused our research to combine and produce a series of 3-hydroxy-6-methyl-2-[(substitutedpiperidine-1-
yl)methyl]-4H-pyran-4-one (compounds 7a-t) structured compounds. This series is composed of a total of
2
1,22
1
9 compounds; 6 of which have previously been described (compounds 7a, 7c, 7d, 7f, 7p, 7s),
while
our group has synthesized 13 of them for the first time (Figures 1 and 2). All these compounds have been
1
characterized in our laboratory with physicochemical, spectral (IR, H-NMR), and elemental analysis data.
4
48

Mannich base derivatives of 3-hydroxy-6-methyl-4H-pyran-4-one..., D. US, et al.,
O
O
N
O
O
N
N
(
1)
(2)
O
(3)
O
O
O
OH
OH
OH
HO
O
H C
3
O
H C
3
N
R
(7a-t)
(4)
(6)
Figure 1. Structures of the compounds.
Further, the broth dilution method was used to determine minimum inhibitory concentration (MIC), and
to establish their antituberculostatic and antimicrobial activities over M. smegmatis, E. coli, S. paratyphi, S.
flexneri, and E. gergoviae (Table). This series of compounds was chosen to constitute a homolog series as they
were also screened for their DNA gyrase enzyme inhibition to determine their activity using a DNA gyrase
supercoiling assay.
O
O
O
O
H N
OH
OH
OH
OH
(a)
(b)
R
HO
Cl
O
O
H3C
O
(c)
H3C
O
N
(5)
(6)
(7a-t)
R
(4)
o
Reagents and conditions; (a) SOCl , 2h, rt ; (b) Zn/HCl,H O, 3h, 70 C; (c) HCHO, MeOH
2
2
Figure 2. General synthesis of the compounds.
Experimental
All reagents were obtained from commercial sources. Solvents were dried and purified with known conventional
methods. DNA gyrase assay kit 3 (1000U-K0003) and DNA gyrase enzyme (100U-G1001) were purchased
from Inspiralis Co. (Norwich, UK). Melting points were detected with a Mettler-Toledo FP-62 melting point
apparatus (Columbus, OH, USA) and are uncorrected. IR spectra (KBr) were recorded on a Perkin Elmer
1
1
720X FT-IR spectrometer (Beaconsfield, UK). H-NMR spectra were obtained by Varian Mercury 400, 400
MHz High Performance Digital FT-NMR using DMSO-d6 and tetramethylsilane as internal standard. All
chemical shift values were recorded as δ (ppm). The purity of the compounds was measured by thin-layer
chromatography on silica gel-coated aluminum sheets (Merck, 1.005554, silica gel HF254–361, Type 60, 0.25
mm; Darmstadt, Germany). The elemental analyses of the compounds were performed on a Leco CHNS
9
32 analyzer (Leco Corp., MI, USA). Elemental analysis for C, H, and N were within ± 0.4% of theoretical
4
49

Mannich base derivatives of 3-hydroxy-6-methyl-4H-pyran-4-one..., D. US, et al.,
1
values. H-NMR spectra and elemental analysis were performed at the Central Analysis Laboratory of Ankara
University, Faculty of Pharmacy, in Ankara, Turkey.
General method for synthesis of 2-(chloromethyl)-5-hydroxy-4H-pyran-4-one (5)
Kojic acid (142 g, 2 mol) (4) was dissolved in thionyl chloride (237 g, 2 mol), followed by stirring for 2 h at
room temperature. The yellow solid was filtered, and then washed with cold petroleum ether. Recrystallization
◦
29
◦
from water gave light-yellow crystals (115 g, 72%), melting point (mp) 146-147 C (lit. value 146-147 C).
General method for synthesis of 5-hydroxy-2-methyl-4H-pyran-4-one (6)
Compound 5 (20 g, 0.12 mol) was suspended in water (500 mL). The temperature of the reaction
◦
◦
mixture was raised to 50 C. Zinc dust (16 g, 0.24 mol) was added and stirred at 70 C for 0.5 h. Concentrated
◦
hydrochloric acid (13.6 g, 3 mol) was added dropwise followed by stirring for 4 h at 70-80 C. The solution was
filtered, poured into ice-water and extracted with dichloromethane, dried with anhydrous sodium sulfate, and
evaporated to dryness. Recrystallization of the resulting yellow solid from isopropanol provided compound 6
◦
29
◦
as light-yellow needles (10.2 g, 64%), mp 125-127 C (lit. value 125-127 C).
General method for synthesis of 3-hydroxy-6-methyl-2-[(substitutedpiperidine-1-yl)methyl]-
4
H -pyran-4-one derivatives (7a-t)
A mixture of 0.01 mol of substituted piperidine and 0.01 mol of compound 6 in 20 mL of methanol with
0
.012 mol of 37% formalin was stirred at room temperature until a solid mass precipitated. The compound was
filtered and washed with cold water, dried under vacuum, and recrystallized from suitable solvents.
3
-Hydroxy-6-methyl-2-[(2-methylpiperidine-1-yl)methyl]-4H-pyran-4-one (7b)
−
1
1
Recrystallized from methanol/diethyl ether. IR (KBr) (υ, cm , stretching) 1618 (C=O); H-NMR δ
(ppm), 6.2 (s, 1H), 3.55 (s, 2H), 3.1 (d, 1H), 2.7 (m, 2H), 2.3 (s, 3H), 1.8, 1.7 and 1.5 (m, 6H), 1.05 (m, 3H,
piperidine -CH3). Anal. Calcd. For C13 H19 NO3 : C 65.8, H 8.07, N 5.90. Found: C 66.2, H 8.10, N 5.96. mp
◦
1
48-149 C.
2
-[(2,6-Dimethylpiperidine-1-yl)methyl]-3-hydroxy-6-methyl-4H-pyran-4-one (7e)
−
1
1
Recrystallized from methanol/diethyl ether. IR (KBr) (υ, cm , stretching), 1607 (C=O); H-NMR δ
ppm), 6.2 (s, 1H), 3.50 (s, 2H), 2.6 (m, 2H), 2.3 (s, 3H), 1.75 (m, 1H), 1.6 (m, 2H), 1.35 (m, 1H), 1.1 (m, 6H,
(
piperidine -CH3), 1.0 (m, 2H). Anal. Calcd. For C14 H21 NO3 -0.1HOH: C 66.43, H 8.44, N 5.53. Found: C
◦
6
6.54, H 8.44, N 5.54. mp 156-157 C.
2
-[(5-Ethyl-2-methylpiperidine-1-yl)methyl]-3-hydroxy-6-methyl-4H-pyran-4-one (7g)
−
1
1
Recrystallized from methanol/diethyl ether. IR (KBr) (υ, cm , stretching), 1618 (C=O); H-NMR δ
ppm), 6.2 (s, 1H), 3.5 (s, 2H), 3.0 (m, 1H), 2.4 (s, 3H), 1.9-1.2 (m, 10H), 0.9 (m, 5H, piperidine-C2 H5 and
(
-
CH3). Anal. Calcd. For C15 H23 NO3 -0.25HOH: C 66.76, H 8.78, N 5.19. Found: C 66.65, H 8.90, N 5.14.
◦
mp 144-145 C.
3
-Hydroxy-6-methyl-2-[(2-propylpiperidine-1-yl)methyl]-4H-pyran-4-one (7h)
−
1
1
Recrystallized from methanol/diethyl ether. IR (KBr) (υ, cm , stretching), 1605 (C=O); H-NMR
δ (ppm), 6.2 (s, 1H), 3.50 (s, 2H), 2.95 (m, 1H), 2.45 (m, 2H), 2.25 (s, 3H), 1.8-1.2 (m, 10H, piperidine and
4
50

Mannich base derivatives of 3-hydroxy-6-methyl-4H-pyran-4-one..., D. US, et al.,
-
C3 H7), 0.9 (t, 3H, propyl). Anal. Calcd. For C15 H23 NO3 -0.4HOH: C 66.10, H 8.80, N 5.14. Found: C 66.01,
◦
H 9.20, N 5.09. mp 165-166 C.
3
-Hydroxy-6-methyl-2-[(4-propylpiperidine-1-yl)methyl]-4H-pyran-4-one (7i)
−
1
1
Recrystallized from methanol/diethyl ether. IR (KBr) (υ, cm , stretching), 1612 (C=O); H-NMR δ
(ppm), 6.2 (s, 1H), 3.6 (s, 2H), 3.0 (d, 2H), 2.3 (s, 3H), 2.15 (t, 2H), 1.7 (d, 2H), 1.4-1.1 (m, 7H, piperidine
and -C3 H7), 0.9 (t, 3H, -C3 H7). Anal. Calcd. For C15 H23 NO3 -0.1HOH: C 67.44, H 8.75, N 5.24. Found: C
◦
6
7.42 H 8.76, N 5.16. mp 157-158 C.
3
-Hydroxy-2-[(3-hydroxypiperidine-1-yl)methyl]-6-methyl-4H-pyran-4-one (7j)
−
1
1
Recrystallized from methanol/diethyl ether. IR (KBr) (υ, cm , stretching), 1608 (C=O); H-NMR δ
(
ppm), 6.2 (s, 1H), 3.85 (m, 1H), 3.65 (s, 2H), 2.7-2.5 (m, 4H), 2.30 (s, 3H), 1.9-1.5 (m, 4H). Anal. Calcd. For
◦
C12 H17 NO4 : C 60.24, H 7.16, N 5.85. Found: C 59.91, H 7.32, N 5.80. mp 136-137 C.
3
-Hydroxy-2-[(4-hydroxypiperidine-1-yl)methyl]-6-methyl-4H-pyran-4-one (7k)
−
1
1
Recrystallized from methanol/diethyl ether. IR (KBr) (υ, cm , stretching) 1612 (C=O); H-NMR δ
(ppm), 6.2 (s, 1H), 3.50 (s, 2H), 3.45 (m, 1H), 2.7 (m, 2H), 2.25 (s, 3H), 2.15 (m, 2H), 1.7 and 1.4 (m, 4H).
Anal. Calcd. For C12 H17 NO4 -0.25HOH: C 59.12, H 7.24, N 5.75. Found: C 58.97, H 7.07, N 5.70. mp 155-156
◦
C.
3
-Hydroxy-2-{[3-(hydroxymethyl)piperidine-1-yl]methyl}-6-methyl-4H-pyran-4-one (7l)
1 1
−
Recrystallized from methanol/diethyl ether. IR (KBr) (υ, cm , stretching), 1618 (C=O); H-NMR δ
ppm), 6.2 (s, 1H), 3.65 (s, 2H), 3.5 (m, 2H, methanol CH2), 3.0-2.85 (m, 2H), 2.3 (s, 3H), 2.15-1.6 (m, 6H),
(
1
1
.1 (m, 1H). Anal. Calcd. For C13 H19 NO4 : C 61.64, H 7.56, N 5.53. Found: C 61.71, H 7.87, N 5.52. mp
◦
47-148 C.
3
-Hydroxy-2-{[4-(hydroxymethyl)piperidine-1-yl]methyl}-6-methyl-4H-pyran-4-one (7m)
1 1
−
Recrystallized from methanol/diethyl ether. IR (KBr) (υ, cm , stretching), 1626 (C=O); H-NMR δ
ppm), 6.2 (s, 1H), 3.60 (s, 2H), 3.50 (d, 2H, methanol CH2), 3.0 (d, 2H), 2.3 (s, 3H), 2.2 (t, 2H), 1.73-1.63 (m,
(
4
1
H). Anal. Calcd. For C13 H19 NO4 -0.5HOH: C 59.53, H 7.69, N 5.34. Found: C 59.34, H 7.98, N 5.30. mp
◦
52-153 C.
3
-Hydroxy-6-methyl-2-[(4-phenylpiperidine-1-yl)methyl]-4H-pyran-4-one (7n)
−
1
1
Recrystallized from methanol. IR (KBr) (υ, cm , stretching), 1614 (C=O); H-NMR δ (ppm), 7.3-7.1
(
m, 5H, aromatic CH), 6.1 (s, 1H), 3.55 (s, 2H), 2.95 (d, 3H), 2.3 (s, 3H), 1.6 and 1.8 (m, 4H). Anal. Calcd.
◦
For C 18 H21 NO3 : C 72.22, H 7.07, N 4.68. Found: C 72.03, H 6.89, N 4.72. mp 167-168 C.
3
-Hydroxy-2-{[4-(4-hydroxyphenyl)piperidine-1-yl]methyl}-6-methyl-4H-pyran-4-one (7o)
1 1
−
Recrystallized from methanol. IR (KBr) (υ, cm , stretching), 1615 (C=O); H-NMR δ (ppm), 7.5-7.3
(m, 4H, aromatic CH), 6.1 (s, 1H), 4.88 (s, 1H, Ar-OH), 3.55 (s, 2H), 2.5-2.7 (m, 5H), 2.25 (s, 3H), 1.9 and
1
1
.6 (m, 4H). Anal. Calcd. For C18 H21 NO4 : C 68.55, H 6.71, N 4.44. Found: C 68.35, H 6.54, N 4.49. mp
◦
77-178 C.
4
51

Mannich base derivatives of 3-hydroxy-6-methyl-4H-pyran-4-one..., D. US, et al.,
3
-Hydroxy-6-methyl-2-{[4-(morpholin-4-yl)piperidine-1-yl]methyl}-4H-pyran-4-one (7r)
−
1
1
Recrystallized from chloroform/petroleum ether. IR (KBr) (υ, cm , stretching), 1620 (C=O); H-
NMR δ (ppm), 6.2 (s, 1H), 3.55 (t, 4H, morpholine CH2), 3.45 (s, 2H), 2.85 (m, 2H), 2.4 (t, 4H, morpholine
CH2), 2.25 (s, 3H), 2.05 (m, 3H), 1.7 and 1.35 (m, 4H). Anal. Calcd. For C16 H24 N2 O4 -0.2HOH: C 61.60, H
◦
7
.88, N 8.98. Found: C 61.54, H 7.49, N 8.82. mp 157-158 C.
Ethyl 1-[(3-hydroxy-6-methyl-4-oxo-4H-pyran-2-yl)methyl]piperidine-4-carboxylate (7t)
−
1
Recrystallized from chloroform/petroleum ether. IR (KBr) (υ, cm , stretching), 1726 (C=O), 1616
1
(
C=O); H-NMR δ (ppm), 6.1 (s, 1H), 4.05 (q, 2H), 3.5 (s, 2H), 2.8 (m, 2H), 2.3 (m, 1H), 2.25 (s, 3H), 2.15
(
m, 2H), 1.8 and 1.55 (m, 4H), 1.2 (t, 3H). Anal. Calcd. For C15 H21 NO5 -0.1HOH: C 60.54, H 7.2, N 4.71.
◦
Found: C 60.56, H 6.87, N 4.73. mp 127-128 C.
Microbiology
Broth Dilution Method for Minimum Inhibitory Concentration (MIC)
MICs of compounds prepared in tubes were determined by broth dilution using Middlebrook 7H10 agar
supplemented with 10% oleic acid-albumin-dextrose-catalase for mycobacteria and Tryptic Soy Broth for other
species. The tested dilutions ranged from 128 to 0.5 μg/mL using dimethyl sulfoxide (DMSO) as solvent for all
compounds. Mycobacteria were suspended in Middlebrook 7H10 broth and the others in Tryptic Soy Broth to
8
match the turbidity of 1 McFarland standard (2 × 10 cfu/mL). The tube slants were inoculated with undiluted
◦
or 1/100 diluted bacterial suspensions and incubated at 37 C. The slants were examined until visible colonies
were seen in the control tube. The controls prepared with the amounts of DMSO used in the dilutions did
not show any inhibitory activity under these circumstances. The MIC value of each isolate was the lowest
concentration of the compound that inhibited visible bacterial growth.
DNA gyrase supercoiling assay
DNA gyrase supercoiling assays were performed with a gyrase supercoiling assay kit (Inspiralis) according to
the manufacturer’s instructions and analyzed by monitoring the conversion of relaxed pBR322 plasmid to its
supercoiled form using DNA gel electrophoresis. Essentially, 1 U E. coli DNA gyrase was first diluted in 5×
gyrase buffer and incubated in an assay buffer (35 mM Tris.HCl (pH 7.5), 24 mM KCl, 4 mM MgCl2 , 2 mM
DTT, 1.8 mM Spermidine, 1 mM ATP, 6.5% (w/v) glycerol, and 0.1 mg/mL BSA), with 0.5 μg of pBR322
◦
plasmid and a series of synthesized compound dilutions at 37 C for 30 min. The reactions were stopped by
the addition of Stop Dye (40% sucrose, 100 mM Tris.HCl (pH 7.5), 1 mM EDTA, 0.5 mg/mL bromophenol
blue) and loaded on a TAE agarose gel (1%). Gels were visualized using a gel documentation system (BIORAD
Chemi Doc).
Since high levels of DMSO are known to also affect DNA gyrase activity, titration was used to determine
the minimum amount of DMSO to be used in the assays and 5% DMSO (with negligible or no effect on the
gyrase) was chosen to dilute the compounds (data not shown).
4
52

Mannich base derivatives of 3-hydroxy-6-methyl-4H-pyran-4-one..., D. US, et al.,
Result and discussion
Final compounds that we synthesized were as follows: kojic acid (compound 4) was reacted with thionyl chloride
1
2
at room temperature to achieve compound 5. Subsequently, this intermediate (compound 5) was reduced to
◦
allomaltol (compound 6) using zinc dust suspension in water and concentrated hydrochloric acid at 70-80
C
1
2,20−22,30
over 4.5 h. This reaction exclusively affects methyl chlorine and leaves the carbonyl group likewise.
After purification, to conclude, allomaltol was reacted with suitable piperidine derivatives in methanol/formalin
medium until the formation of desired 3-hydroxy-6-methyl-2-[(substitutedpiperidine-1-yl)methyl]-4H-pyran-4-
one derivatives (compounds 7a-t) as precipitates. In the Mannich reaction; substitution for the second position
of the ring system is favored due to the existence of a free enolic hydroxyl group in the third position for this
2
6
ring system. Compounds were crystallized from appropriate solvents in moderate yields (Figure 2).
Table. MIC (μg/mL) of the compounds over E. coli, S. paratyphi, S. flexneri, E. gergoviae, and M. smegmatis.
O
OH
H C
3
O
N
R
R
E. coli S. paratyphi S. flexneri E. gergoviae M. smegmatis
7
7
a
hydrogen
32
64
128
64
32
64
128
32
32
64
32
32
64
32
64
64
64
32
64
128
128
128
128
64
128
64
128
64
16
16
8
b
2-methyl
7
c
3-methyl
128
128
64
128
64
7
d
4-methyl
8
7
e
2,6-dimethyl
3,5-dimethyl
5-ethyl-2-methyl
2-propyl
64
8
7
f
32
64
64
8
7
7
g
128
64
64
128
64
16
16
8
h
128
128
64
7
7
i
4-propyl
64
64
j
3-hydroxy
32
128
128
64
8
7
k
4-hydroxy
32
64
16
16
16
8
7
l
3-(hydroxymethyl)
4-(hydroxymethyl)
4-phenyl
64
32
7
m
32
32
64
7
n
o
p
128
64
64
128
64
7
4-(4-hydroxyphenyl)
4-piperidin-1-yl
4-(4-morpholino)
4-benzyl
64
8
7
32
64
64
16
16
16
8
7
7
7
r
s
t
64
64
64
32
64
64
4-ethoxycarbonyl
128
64
64
1
1
IR, H-NMR, and elemental analysis characterized the proposed structure. In the H-NMR spectra of
compounds, piperidine ring protons are dispersed between δ 1.1 to 3.45 ppm in various integrals according
4
53

Mannich base derivatives of 3-hydroxy-6-methyl-4H-pyran-4-one..., D. US, et al.,
to their position toward ring nitrogen and their neighboring replacements of different electronic environments.
Vinylic protons of the pyran ring system are observed as singlets at δ 6.1-6.2 ppm. The methylene linkage
protons between pyran and piperidine rings occur between δ 3.45 and 3.55 ppm as singlets. The deletion of
a singlet around 9-9.5 ppm belonging to the proton at the sixth position of the allomaltol structure and the
formation of these new singlets confirmed the desired 3-hydroxy-6-methyl-2-[(substitutedpiperidine-1-yl)methyl]-
4
H -pyran-4-one structures. Spectral data of 6 compounds (compounds 7a, 7c, 7d, 7f, 7p, 7s) are compatible
2
1,22
with the data previously described for those.
All the other protons were seen according to the expected
2
0−22,29
chemical shift and integral values.
Biological evaluation
Every compound belonging to the series was tested over E. coli, S. paratyphi, S. flexneri, and E. gergoviae as
gram-negative and M. smegmatis as gram-positive type bacteria for their MIC values. All 3-hydroxy-6-methyl-
2
-[(substitutedpiperidine-1-yl)methyl]-4H -pyran-4-one derivatives showed serious inhibitory activity over M.
smegmatis growth in concentrations of 8-16 μg/mL, showing moderate to minimum activity over other bacteria
types. What distinguishes Mycobacteria from the other species tested is that, although Mycobacterium species
can still be considered gram-positive, they are not truly gram-positive in the traditional sense, and indeed
1
7
some researchers classify them as acid-fast gram-positive bacteria
and thus the growth inhibitory effect
could be due to DNA gyrase activity and/or topoisomerase activity inhibition by these or similar compounds.
Moreover, Mycobacterium species share a characteristic cell wall, thicker than that in many other bacteria, with
7
a hydrophobic character.
These results indicated that these compounds were selectively effective over gram-positive bacteria either
by their high lipophilic properties or by their selective DNA gyrase and topoisomerases enzyme inhibitor
activities like quinolones (1), naphthyridones (2), and coumarins (3).
In this work, we focused on the DNA gyrase inhibitory effect of these compounds to explain the significance
of the MIC. DNA gyrase is a topoisomerase, which can convert relaxed pBR322 plasmid to its supercoiled
topoisomer; therefore DNA gyrase activity can be monitored as 2 bands on an agarose gel. The upper band
is open-circular (nicked) plasmid, whereas the faster running band is negatively supercoiled (closed circular)
plasmid. Using this assay, we can analyze the effect of various compounds on DNA gyrase activity.
When the 3-hydroxy-6-methyl-2-[(substitutedpiperidine-1-yl)methyl]-4H-pyran-4-one series of compounds
were analyzed for any effect on the supercoiling activity of DNA gyrase, we observed a minimal effect. For most
compounds, even in increasing amounts, did not inhibit the supercoiling activity of DNA gyrase. Only a few
compounds (compounds 7a, c, f, and n) showed activity over this enzyme to a certain extent (Figure 3).
The fact that they did not have more than a mild inhibitory effect on DNA gyrase enzyme, however, does
not rule out the possibility that they may still inhibit bacterial growth through interfering with the function
of other topoisomerases. Indeed, topoisomerase IV is the subtype commonly found in many gram-positive
bacteria and has a specialized function in mediating the decatenation of interlocked daughter chromosome
during replication. However, it should be noted that, for Mycobacterium in particular, there are not even any
1
0
topoisomerase IV genes, but instead topoisomerase II.
Our current research effort is focused on studying
the inhibitory role of these compounds on topoisomerase II, and in particular on their effects over multi-drug
resistant and nonresistant Mycobacterium species, especially Mycobacterium tuberculosis.
4
54

Mannich base derivatives of 3-hydroxy-6-methyl-4H-pyran-4-one..., D. US, et al.,
Figure 3. Effect of some 3-hydroxy-6-methyl-2-[(substitutedpiperidine-1-yl)methyl]-4H -pyran-4-one derivatives on
DNA gyrase supercoiling activity. The bands in the agarose gel pictures indicate the topoisomers of pBR322 plasmid.
OC and S indicate open circular and supercoiled pBR322 plasmid DNA, respectively. Empty vehicle control; compounds
are titrated at 2, 4, 8, 16, 32, 64 μg/mL concentration). Most compounds showed either no effect on DNA gyrase
activity, or a slight increase. Only these compounds indicated (*) show minor inhibition of DNA gyrase activity, where
some non-effective ones were also given as examples.
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