Synthesis, physico-chemical properties, and antimicrobial evaluation of a new series of iron(III) hexadentate chelators

Article abstract of DOI:10.1007/s00044-012-0229-1

A series of related 3-hydroxypyridin-4-one hexadentate ligands have been synthesized. These chelators were found to possess a high affinity for iron(III), with a pFe value of about 30. As iron is a critical element to the survival of bacteria, these chelators were predicted to inhibit the growth of bacteria by disrupting bacterial iron absorption. Indeed, they were demonstrated to possess appreciable inhibitory activity against both Gram-positive and Gram-negative bacteria, and therefore, they have potential as antimicrobial agents. 1c and 1g were found to be particularly effective against Gram-negative species. Graphical Abstract: A series of related 3-hydroxypyridin-4-one hexadentate ligands have been synthesized. These chelators were found to possess a high affinity for iron(III), and exhibit appreciable inhibitory activity against both Gram-positive and Gram-negative bacteria, and therefore, they have potential as antimicrobial agents. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-012-0229-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:2351–2359
DOI 10.1007/s00044-012-0229-1
ORIGINAL RESEARCH
Synthesis, physico-chemical properties, and antimicrobial
evaluation of a new series of iron(III) hexadentate chelators
•
Yuan-Yuan Xie Mu-Song Liu Pan-Pan Hu
•
•
•
Xiao-Le Kong Di-Hong Qiu Ji-Lin Xu
•
•
•
Robert C. Hider Tao Zhou
Received: 22 December 2011 / Accepted: 11 September 2012 / Published online: 22 September 2012
Ó Springer Science+Business Media, LLC 2012
Abstract A series of related 3-hydroxypyridin-4-one
hexadentate ligands have been synthesized. These chelators
were found to possess a high affinity for iron(III), with a
pFe value of about 30. As iron is a critical element to the
survival of bacteria, these chelators were predicted to
inhibit the growth of bacteria by disrupting bacterial iron
absorption. Indeed, they were demonstrated to possess
appreciable inhibitory activity against both Gram-positive
and Gram-negative bacteria, and therefore, they have
potential as antimicrobial agents. 1c and 1g were found to
be particularly effective against Gram-negative species.
Introduction
Bacterial and fungal resistance to antimicrobial agents is a
growing problem worldwide (Nikaido and Zgurskaya,
1999; Mitscher et al., 1999; Hogan and Kolter, 2002).
Consequently, there is an urgent need for the development
of novel types of antimicrobial agents targeting unique
mechanisms and pathways. Iron is an essential element for
the growth of virtually all bacteria and fungi. Thus, limiting
the iron absorption of microorganism should in principle
be an effective strategy to inhibit microbial growth.
Most microorganisms have developed efficient methods of
absorbing iron from the environment and many microor-
ganisms secrete siderophores to scavenge iron (Lewin,
1984; Hider and Kong, 2010). In principle, such uptake can
be interrupted by the introduction of high affinity iron
selective chelating agents (Lowe and Phillips, 1962;
Bergan et al., 2001). The iron affinity of these agents must
be extraordinally high, so that they can efficiently out-
compete siderophores. Furthermore, the structure of che-
lators should differ appreciably from those of siderophores,
otherwise the iron-chelator complex will be able to supply
iron to the microorganism via the iron-siderophore trans-
porter. Thus, iron(III)-selective hexadentate chelators with
a high affinity for iron are predicted to inhibit the growth of
a wide range of bacteria. We have previously demonstrated
that compound 1a (Fig. 1) and other two compounds,
which have the structure of a hexadentate 3-hydroxypyri-
din-4-one, inhibit the growth of both Gram-positive and
Gram-negative bacteria (Qiu et al., 2011; Xu et al., 2011).
Further more, it has also been demonstrated that the iron
complex of 1a cannot be utilized by a range of bacteria,
including Staphylococcus aureus and Escherichia coli
(Zhou et al., 2011). However, some chelators with high
iron-binding constants, for instance, ethylenediamine-N,
Keywords Iron chelator ꢀ 3-Hydroxypyridin-4-one ꢀ
Hexadentate ꢀ Antimicrobial activity
Y.-Y. Xie ꢀ M.-S. Liu
College of Pharmaceutical Sciences,
Zhejiang University of Technology,
Hangzhou 310014, People’s Republic of China
P.-P. Hu ꢀ T. Zhou (&)
School of Food Science and Biotechnology,
Zhejiang Gongshang University, Hangzhou,
Zhejiang 310035, People’s Republic of China
e-mail: taozhou@zjgsu.edu.cn
X.-L. Kong ꢀ R. C. Hider
Division of Pharmaceutical Science,
King’s College London, Franklin–Wilkins Building,
150 Stamford Street, London SE1 9NH, UK
D.-H. Qiu ꢀ J.-L. Xu
Faculty of Life Science and Biotechnology,
Ningbo University, Ningbo, Zhejiang 315211,
People’s Republic of China
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Med Chem Res (2013) 22:2351–2359
O
over anhydrous sodium sulfate. After removal of the solvent,
the residue was purified byrecrystallization from diethyl ether
to obtain the product as a white solid (14.75 g, 68.12 mmol).
Yield: 85.8 %; 1H NMR (CDCl3) d 2.08 (s, 3H, CH3), 5.15 (s,
2H, ArCH2), 6.35 (d, J = 5.6 Hz, 1H, Pyridinone C5-H),
7.31–7.39 (m, 5H, Ar), 7.58 (d, J = 5.6 Hz, 1H, Pyridinone
C6-H). ESI–MS m/z 217 ([M ? H]?).
N
O
HO
H
HN
H
N
O
N
N
H
OH
NH OH
O
O
O
N
H
3-(Benzyloxy)-4-oxo-4H-pyran-2-carbadehyde (4)
1a
A mixture of 3 (5 g, 23.15 mmol) and SeO2 (7.1 g,
69.4 mmol) in bromobenzene (80 mL) was stirred vigor-
ously at 159 °C for 14 h. The reaction mixture was cooled to
room temperature and filtered, the filtrate was concentrated
under vacuum. The residue was purified by chromatography
(SiO2; EtOAc/hexane 1:1) to give the product as a brown oil
(4.15 g, 18.06 mmol). Yield: 78 %; 1H NMR (CDCl3) d 5.46
(s, 2H, ArCH2), 6.47 (d, J = 5.6 Hz, 1H, Pyridinone C5-H),
7.32 (m, 5H, Ar), 7.72 (d, J = 5.6 Hz, 1H, Pyridinone C6-
H), 9.83 (s, 1H, CHO), and ESI–MS m/z 231 ([M ? H]?).
Fig. 1 Structure of hexadentate hydroxypyridinone 1a
N0-bis(2-hydroxyphenyl) acetic acid (EDHPA, logK 34),
N,N0-bis(2-hydroxybenzyl) ethylenediamine-N,N0-diacetic
acid (HEBD, logK 40), and desferrioxamine (logK 30.4)
(Anderson, 1980) have been reported to only possess a
weak ability to inhibit Gram-positive and Gram-negative
bacterial growth (Chew et al., 1985), indicating that iron
affinity of the chelator is not the only factor which affects
the antimicrobial potential of an iron chelator. In an
attempt to investigate the influence of hydrophobicity on
the antimicrobial efficacy of hexadentate hydroxypyridi-
nones, we synthesized a range of 1a analog possessing
different oil–water partition coefficients.
3-(Benzyloxy)-4-oxo-4H-pyran-2-carboxylic acid (5)
To a mixture of4(4.60 g,20 mmol)inacetoneandwater(1:1)
was added sodium hypochlorite (2.18 g, 24 mmol) and sulf-
anic acid (2.92 g, 30 mmol) with vigorous stirring. The stir-
ring was continued at room temperature for 2 h. The
precipitate was collected by filtration, washed with acetone
and dried in vacuo to give product 5 as a white solid (4.77 g,
Experimental section
1
General procedures
19.4 mmol). Yield: 97 %; m.p. 150.1–151.8 °C. H NMR
(DMSO-d6) d 5.10 (s, 2H, CH2), 6.53 (d, J = 5.6 Hz, 1H,
Pyridinone C5-H), 7.44–7.41 (m, 5H, Ar), 8.19 (d, J =
5.6 Hz, 1H, PyridinoneC6-H). ESI–MSm/z247([M ? H]?).
All chemicals were of AR grade and used without any
further purification. Melting points were determined using
an SGW X-4A Digital Melting Point Apparatus and were
uncorrected. 1H NMR and 13C NMR spectra were recorded
on a Bruker Avance 400 or a Bruker Avance 500 spec-
trometer with TMS as an internal standard. Electrospray
ionization (ESI) mass spectra were obtained by infusing
samples into an LCQ Deca XP ion trap instrument. High
resolution mass spectra (HRMS) were determined on
Waters QTOF micro.
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl) tris(3-
methyl-4-oxo-4H-pyran-2-carboxamide) (7)
The mixture of compound 5 (4 g, 16.26 mmol), N-hydroxy-
succinmide (NHS, 1.87 g, 16.26 mmol) in THF (70 mL) was
stirred at room temperature for 0.5 h, N,N0-dicyclohexylcar-
bodiimide (DCC, 2.35 g, 16.26 mmol) was then added. The
stirring was continued for 3 h, N1,N1-bis(2-aminoethyl)eth-
ane-1,2-diamine (0.66 g, 4.52 mmol) in THF (10 mL) was
added dropwise. The reaction mixture was stirred at room
temperature overnight, and the formed precipitate was filtered
off and washed with THF (20 mL). The filtrate was concen-
trated under vacuum to give a yellow oil which was then
dissolved in CH2Cl2 (100 mL), washed with 0.5 % sodium
hydroxide (2 9 40 mL) and brine successively, dried over
anhydrous sodium sulfate, purified by chromatography (SiO2,
CH2Cl2/CH3OH, 10:1), giving the product 7 as a pale yellow
powder (2.17 g, 2.62 mmol). Yield: 58 %. 1H NMR (CDCl3)
d 2.29 (t, J = 6.0 Hz, 6H, CH2), 3.11 (t, J = 6.0 Hz, 6H,
3-(Benzyloxy)-2-methyl-4H-pyran-4-one (3)
To a solution of 2 (10 g, 79.4 mmol) in methanol (150 mL)
was added 35 % sodium hydroxide (10 mL) dropwise with
stirring vigorous at 80 °C. Benzyl chloride was then added
dropwise to the above mixture with stirring over 30 min
and refluxed for 8 h. After cooled and filtered, the filtrate
was concentrated on a rotatory evaporator to remove most
of the organic solvent, and extracted with dichloromethane
(3 9 50 mL). The combined organic extracts were washed
with 5 % sodium hydroxide (2 9 50 mL) and brine, dried
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CH2), 5.34 (s, CH2, 6H), 6.47 (d, J = 5.6 Hz, 3H, Pyridinone
C5-H), 7.35–7.33 (m, 15H, Ph), 7.69 (s, 3H, NH), and 7.82 (d,
J = 5.6 Hz, 3H, Pyridinone C6-H). ESI–MS m/z 831
([M ? H]?).
(t, J = 6.0 Hz, 6H, CH2,) 2.88 (t, 6H, J = 6.0 Hz, CH2),
3.73 (t, J = 7.2 Hz, 6H, CH2), 5.01 (s, 6H, CH2), 6.30
(d, J = 7.2 Hz, 3H, Pyridinone C5-H), 7.15 (d,
J = 7.2 Hz, 3H, Pyridinone C6-H), 7.30 (m, 9H, Ph), 7.44
(m, 6H, Ph). ESI–MS m/z 996 ([M ? H]?).
General procedure for the synthesis of N,N0,
N00-(2,20,200-nitrilotris(ethane-2,1,diyl)tris(3-
(benzyloxy)-4-oxo-4H-pyran-2-carboxamide)
derivatives (8a–i)
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris
(3-(benzyloxy)-1-hexyl-4-oxo-1,4-dihydropyridine-
2-carboxamide) (8e)
A mixture of compound 7 (0.50 g, 0.602 mmol) and vari-
ous amines (2.166 mmol) in ethanol was refluxed for 3 h.
After removal of the solvent, the residual brown oil was
purified by chromatography (SiO2) using CH2Cl2/CH3OH
as an eluent to provide product 8.
1
Yield: 32 %. H NMR(CDCl3) d 0.89 (t, J = 6.8 Hz, 9H,
CH3), 1.28 (m, 12H, CH2), 1.76 (m, 6H, CH2), 1.85 (m, 6H,
CH2), 2.14 (t, J = 6.4 Hz, 6H, CH2), 2.84 (t, J = 6.4 Hz,
6H, CH2), 3.74 (t, J = 6.4 Hz, 6H, CH2), 5.02 (s, 6H,
CH2), 6.27 (d, J = 7.6 Hz, 3H, Pyridinone C5-H), 7.15
(d, J = 7.6 Hz, 3H, Pyridinone C6-H), 7.29 (m, 9H, Ph),
7.43 (m, 6H, Ph). ESI–MS m/z 1080 ([M ? H]?).
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris
(3-(benzyloxy)-4-oxo-1,4-dihydropyridine-2-carboxamide)
(8a)
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris
(3-(benzyloxy)-1-(2-hydroxyethyl)-4-oxo-1,
4-dihydropyridine-2-carboxamide) (8f)
1
Yield: 57 %. H NMR (CDCl3) d 2.27 (t, J = 6.0 Hz, 6H,
CH2), 3.06 (q, J = 6.0 Hz, 6H, CH2), 5.32 (s, 6H, CH2),
6.46 (d, J = 7.2 Hz, 3H, Pyridinone C5-H), 7.27–7.32 (br,
15H, Ph), 7.54 (d, J = 7.2 Hz, 3H, Pyridinone C6-H),7.95
(t, J = 6.4 Hz, 3H, NH). ESI–MS m/z 828 ([M ? H]?).
1
Yield: 25 %. H NMR (DMSO-d6) d 2.51 (t, J = 6.0 Hz,
6H, CH2), 3.15 (t, J = 6.0 Hz, 6H, CH2), 3.74 (t, J =
5.6 Hz, 6H, CH2), 3.86 (t, J = 5.6 Hz, 6H, CH2), 5.06 (s,
6H, CH2), 6.19 (d, J = 7.2 Hz, 3H, Pyridinone C5-H), 7.31
(d, J = 7.2 Hz, 3H, Pyridinone C6-H), 7.45–7.49 (m, 15H,
Ph). ESI–MS m/z 960 ([M ? H]?).
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris
(3-(benzyloxy)-1-methyl-4-oxo-1,4-dihydropyridine-
2-carboxamide) (8b)
1
Yield: 45 %. H NMR (CDCl3) d 2.14 (t, J = 6.0 Hz, 6H,
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris(3-
(benzyloxy)-1-(2-methoxyethyl)-4-oxo-1,4-
dihydropyridine-2-carboxamide) (8g)
CH2), 2.82 (t, J = 6.0 Hz, 6H, CH2), 3.57 (s, 9H, CH3),
5.05 (s, 6H, CH2), 6.24 (d, J = 7.2 Hz, 3H, Pyridinone
C5-H), 7.14 (d, J = 7.2 Hz, 3H, Pyridinone C6-H), 7.32 (m,
9H, Ph), 7.44 (m, 6H, Ph). ESI–MS m/z 870 ([M ? H]?).
1
Yield: 40 %. H NMR (CDCl3) d 2.11 (t, J = 6.0 Hz, 6H,
CH2), 2.82 (t, J = 6.0 Hz, 6H, CH2), 3.31 (s, 9H, CH3),
3.61 (t, J = 4.8 Hz, 6H), 3.90 (t, J = 4.8 Hz, 6H, CH2),
5.03 (s, 6H, CH2), 6.24 (d, J = 7.2 Hz, 3H, Pyridinone C5-
H), 7.29 (m, 12H, Ph and buried Pyridinone C6-H), 7.40
(m, 6H, Ph). ESI–MS m/z 1002 ([M ? H]?).
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris
(3-(benzyloxy)-1-ethyl-4-oxo-1,4-dihydropyridine-
2-carboxamide) (8c)
1
Yield: 42 %. H NMR (CDCl3) d 1.38 (t, J = 6.0 Hz, 9H,
CH3), 2.39 (t, J = 6.0 Hz, 6H, CH2), 3.05 (t, J = 6.0 Hz,
6H, CH2), 4.05 (q, J = 6.0 Hz, 6H, CH2), 5.16 (s, 6H,
CH2), 7.14 (d, J = 6.8 Hz, 3H, Pyridinone C5-H), 7.21–
7.32 (m, 15H, Ph), 8.15 (d, J = 6.8 Hz, 3H, Pyridinone
C6-H). ESI–MS m/z 912 ([M ? H]?).
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris
(3-(benzyloxy)-1-(2-hydroxyethoxyl)-4-oxo-1,
4-dihydropyridine-2-carboxamide) (8h)
1
Yield: 25 %. H NMR (DMSO-d6) d 2.67 (t, J = 6.0 Hz,
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris
(3-(benzyloxy)-1-butyl-4-oxo-1,4-dihydropyridine-
2-carboxamide) (8d)
6H, CH2), 3.22 (t, J = 6.0 Hz, 6H, CH2), 3.32 (m, 6H,
CH2), 3.47 (m, 6H, CH2), 3.65 (t, J = 5.2 Hz, 6H, CH2),
3.96 (t, J = 5.2 Hz, 6H, CH2), 5.05 (s, 6H, CH2), 6.21
(d, J = 7.6 Hz, 3H, Pyridinone C5-H), 7.29–7.38 (m, 15H,
Ph), 7.62 (d, J = 7.6 Hz, 3H, Pyridinone C6-H), 8.82
(t, J = 6.0 Hz, 3H, NH). ESI–MS m/z 1092 ([M ? H]?).
1
Yield: 38 %. H NMR (CDCl3) d 0.92 (t, J = 7.2 Hz, 9H,
CH3), 1.28 (m, 6H, CH2), 1.85 (m, 6H, CH2), 2.17
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N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris
(3-(benzyloxy)-1-(2-(2-hydroxyethylamino)ethyl)-
4-oxo-1,4-dihydropyridine-2-carboxamide) (8i)
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris(1-ethyl-
3-hydroxy-4-oxo-1,4-dihydro-pyridine-2-carboxamide)
(1c)
1
Yield: 21 %. H NMR (DMSO-d6) d 2.65 (t, J = 5.6 Hz,
Yield: 91 %; IR (KBr) mmax 3404, 3234, 3048, 2978, 2930,
1652, and 1620 cm-1. 1H NMR (DMSO-d6) d 1.40 (t, J =
7.5 Hz, 9H, CH3), 3.43 (t, J = 7.5 Hz, 6H, CH2), 3.81
(t, J = 7.5 Hz, 6H, CH2), 4.23 (q, J = 7.5 Hz, 6H, CH2),
7.37 (d, J = 7.5 Hz, 3H, Pyridinone C5-H), 8.30 (d, J =
7.5 Hz, 3H, Pyridinone C6-H), 9.65 (t, J = 7.5 Hz, 3H,
NH); 13C NMR (100 MHz, DMSO-d6) d 16.48 (CH3),
34.35 (CH2), 51.38 (CH2), 51.99 (CH2), 112.35 (C-5H in
pyridinone), 135.39 (C-2 in pyridinone), 138.09 (C-6H
in pyridinone), 143.14 (C-3 in pyridinone), 159.50 (C-4 in
pyridinone), 161.99 (CO). ESI–MS m/z 642 ([M ? H]?),
HRMS m/z Calcd. for C30H40N7O9 [M ? H]? 642.2886,
found: 642.2890.
6H, CH2), 2.82 (d, J = 5.6 Hz, 6H, CH2), 3.18 (t, J =
5.6 Hz, 6H, CH2), 3.56 (t, J = 7.0 Hz, 6H, CH2), 3.82 (m,
12H, CH2), 5.03 (s, 6H, CH2), 6.21 (d, J = 7.2 Hz, 3H,
Pyridinone C5-H), 7.16–7.34 (m, 15H, Ph), 7.65 (d, J =
7.2 Hz, 3H, Pyridinone C6-H), 8.59 (t, J = 6.0 Hz, 3H,
NH). ESI–MS m/z 1089 (M ? H?), 1111 (M ? Na?).
General procedures for the preparation of hexadentate (1)
To a suspension of 8 (8a–i) (1 mmol) and concentrated
hydrochloric acid (1 mL) in MeOH (30 mL) was added
5 % Pd/C (0.15 g). Hydrogenation was carried out at 30
psi H2 for 5–6 h. After filtration to remove the catalyst,
the filtrate was concentrated to dryness. The residue
was purified by crystallization from methanol/acetone.
Hydrochlorides of hexadentate 1 were obtained as white
solids.
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris(1-butyl-
3-hydroxy-4-oxo-1,4-dihydro-pyridine-2-carboxamide) (1d)
Yield: 95 %; IR (KBr) mmax 3189, 3029, 2955, 2929, 2872,
1
1635, and 1619 cm-1. H NMR (DMSO-d6) d 0.81 (t, J =
7.5 Hz, 9H, CH3), 1.26 (m, 6H, CH2), 1.75 (m, 6H, CH2), 3.44
(t, J = 8.5 Hz, 6H, CH2), 3.81 (t, J = 8.5 Hz, 6H, CH2), 4.18
(t, J = 8.0 Hz, 6H, CH2), 7.21 (d, J = 9.0 Hz, 3H, Pyridi-
none C5-H), 8.21 (d, J = 9.0 Hz, 3H, Pyridinone C6-H), 9.53
(s, 3H, NH); 13C NMR (100 MHz, DMSO-d6) d 13.84 (CH3),
19.25 (CH2), 32.97 (CH2), 34.93 (CH2), 51.77 (CH2), 56.34
(CH2), 112.45 (C-5H in pyridinone), 134.59 (C-2 in pyridi-
none), 139.11 (C-6H in pyridinone), 144.24 (C-3 in pyridi-
none), 160.34 (C-4 in pyridinone), 163.81 (CO). ESI–MS m/z
726 ([M ? H]?); HRMS m/z Calcd. for C36H52N7O9
[M ? H]? 726.3826, found: 726.3812.
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris(3-hydroxy-
4-oxo-1,4-dihydropyridine-2-carboxamide) (1a)
1
Yield: 80 %; H NMR (DMSO-d6) d 3.41 (t, J = 7.5 Hz,
6H, CH2), 3.79 (t, J = 7.5 Hz, 6H, CH2), 7.02 (d, J =
7.5 Hz, 3H, Pyridinone C5-H), 7.82 (d, J = 7.5 Hz, 3H,
Pyridinone C6-H), 9.38 (br, 3H, NH); 13C NMR (DMSO-
d6) d 34.40 (CONHCH2), 51.69 (NHCH2), 113.05 (C-5H in
pyridinone), 127.56 (C-2 in pyridinone), 136.80 (C-6H
in pyridinone), 147.72 (C-3 in pyridinone), 161.50 (C-4 in
pyridinone), 163.35 (CO). ESI–MS m/z 558 ([M ? H]?).
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris(1-hexyl-
3-hydroxy-4-oxo-1,4-dihydro-pyridine-2-carboxamide)
(1e)
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris(3-hydroxy-
1-methyl-4-oxo-1,4-dihydropyridine-2-carboxamide) (1b)
Yield:86 %;IR(KBr)mmax 3194, 3025, 2967, 2850, 1675, and
1619 cm-1
. d 0.88 (t, J =
1H NMR (DMSO-d6)
7.5 Hz, 9H, CH3), 1.25 (br, 18H, CH2), 1.74 (br, 6H, CH2),
3.42(t, J = 7.0 Hz, 6H, CH2), 3.79(q, J = 7.0 Hz, 6H, CH2),
4.14 (t, J = 7.5 Hz, 6H, CH2), 7.20 (d, J = 7.5 Hz, 3H, Py-
ridinone C5-H), 8.20 (d, J = 7.5 Hz, 3H, Pyridinone C6-H),
9.58 (t, J = 7.0 Hz, 3H, NH); 13C NMR (100 MHz, DMSO-
d6) d 13.82 (CH3), 14.22 (CH2), 19.36 (CH2), 22.27 (CH2),
25.69 (CH2), 31.10 (CH2), 51.88 (CH2), 56.16 (CH2), 111.91
(C-5H in pyridinone), 133.48 (C-2 in pyridinone), 139.08 (C-
6H in pyridinone), 144.41 (C-3 in pyridinone), 160.64 (C-4 in
pyridinone), 164.86 (CO). ESI–MS m/z 810 ([M ? H]?).
HRMS m/z Calcd. for C42H64N7O9 [M ? H]? 810.4764,
found: 810.4756.
Yield: 89 %; IR (KBr) mmax 3387, 3025, 2930, 1671, and
1623 cm-1. 1H NMR (DMSO-d6) d 3.44 (s, 9H, CH3), 3.81
(t, J = 9.0 Hz, 6H, CH2), 3.93 (t, J = 9.0 Hz, 6H, CH2),
7.38 (d, J = 9.0 Hz, 3H, Pyridinone C5-H), 8.22 (d, J =
9.0 Hz, 3H, Pyridinone C6-H), 9.68 (s, 3H, NH); 13C NMR
(DMSO-d6) d 34.54 (CH3), 44.39 (CH2), 51.51 (CH2),
112.33 (C-5H in pyridinone), 136.60 (C-2 in pyridinone),
139.87 (C-6H in pyridinone), 143.87 (C-3 in pyridinone),
159.89 (C-4 in pyridinone), 161.91 (CO). HRMS m/z
Calcd. for C27H34N7O9 [M ? H]? 600.2418, found:
600.2418.
123

Med Chem Res (2013) 22:2351–2359
2355
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris
(3-hydroxy-1-(2-hydroxylethyl)-4-oxo-1,4-dihydro-
pyridine-2-carboxamide) (1f)
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl))tris
(3-hydroxy-1-(2-(2-hydroxyethylamino)ethyl)-4-oxo-
1,4-dihydropyridine-2-carboxamide) (1i)
1
Yield: 83 %; H NMR (DMSO-d6) d 3.25 (t, J = 6.5 Hz,
Yield 86 %; IR (KBr) mmax 3369, 3021, 2961, 2788, 1672,
and 1618 cm-1 1H NMR (DMSO-d6) d 3.25 (t, J =
6H, CH2), 3.43 (t, J = 6.5 Hz, 6H, CH2), 3.71 (t, J =
6.5 Hz, 6H, CH2), 3.91 (t, J = 6.5 Hz, 6H, CH2), 6.31
(d, J = 9.5 Hz, 3H, Pyridinone C5-H), 7.84 (d, J =
9.5 Hz, 3H, Pyridinone C6-H); 13C NMR (DMSO-d6) d
30.76 (CH2), 51.22 (CH2), 56.38 (CH2), 71.61 (CH2),
120.02 (C-5H in pyridinone), 127.67 (C-2 in pyridinone),
139.32 (C-6H in pyridinone), 144.88 (C-3 in pyridinone),
170.19 (C-4 in pyridinone), 173.23 (CO). ESI–MS m/z 690
([M ? H]?); HRMS m/z Calcd. for C30H40N7O12 [M ?
H]? 690.2734, found: 690.2741.
;
4.8 Hz, 6H, CH2), 3.42 (t, J = 6.0 Hz, 6H, CH2), 3.60
(t, J = 4.8 Hz, 6H, CH2), 3.72 (t, J = 5.6 Hz, 6H, CH2),
3.83 (t, J = 5.6 Hz, 6H, CH2), 4.09 (t, J = 5.6 Hz, 6H,
CH2), 7.29 (d, J = 8.0 Hz, 3H, Pyridinone C5-H), 8.10
(d, J = 8.0 Hz, 3H, Pyridinone C6-H), 10.03 (s, 3H, NH);
13C NMR (DMSO-d6) d 39.76 (CH2), 40.09 (CH2), 40.26
(CH2), 49.08 (CH2), 49.90 (CH2), 56.73 (CH2), 112.90
(C-5H in pyridinone), 136.35 (C-2 in pyridinone), 139.78
(C-6H in pyridinone), 148.79 (C-3 in pyridinone), 159.76
(C-4 in pyridinone), 162.36 (CO). ESI–MS m/z 819 ([M ?
H]?). HRMS m/z Calcd. for C36H53N10O12 [M-H]-
817.3843, found 817.3855.
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris
(3-hydroxy-1-(2-methoxyethyl)-4-oxo-1,4-dihydro-
pyridine-2-carboxamide) (1g)
Physico-chemical properties of hexadentate HPOs
Yield: 83 %; IR (KBr) mmax 3385, 3203, 3042, 2950, 2800,
1
1685, 1671, and 1620 cm-1. H NMR (DMSO-d6) d 3.20
pKa determination
(t, J = 6.4 Hz, 6H, CH2), 3.43 (t, J = 6.4 Hz, 6H, CH2),
3.66 (s, 9H, CH3), 3.78 (t, J = 7.2 Hz, 6H, CH2), 4.28
(t, J = 7.2 Hz, 6H, CH2), 7.38 (d, J = 7.2, 6H, Pyridinone
C5-H), 8.15 (d, J = 7.2, 6H, Pyridinone C6-H), 9.71
(s, 3H, NH); 13C NMR (DMSO-d6) d 34.66 (CH2), 51.60
(CH2), 56.40 (CH2), 58.66 (CH2), 70.61 (CH3), 111.94
(C-5H in pyridinone), 136.11 (C-2 in pyridinone), 139.97
(C-6H in pyridinone), 140.01 (C-3 in pyridinone), 159.80
(C-4 in pyridinone), 162.96 (CO). ESI–MS m/z 732 ([M ?
H]?). HRMS m/z Calcd. for C33H46N7O12 [M ? H]?
732.3204, found 732.3228.
The titration system used in this determination comprised
of an autoburette (Metrohm Dosimat 765 l mL syringe)
and a HP 8453 UV–Visible spectrophotometer. 0.1 M KCl
electrolyte solution was used to maintain the ionic strength.
The temperature of the test solutions was maintained in a
thermostatic cuvette holder at 25 0.1 °C using a Cary 1
controller, an argon atmosphere was applied to the entire
titration equipment. The initial sample concentration was
approximately 7 9 10-5M. pKa values were analyzed
from these data by pHab.
Determination of iron(III) affinity
N,N0,N00-(2,20,200-nitrilotris(ethane-2,1-diyl)tris(3-hydroxy-
1-(2-(2-hydroxyethoxy)ethyl)-4-oxo-1,4-dihydro-pyridine-
2-carboxamide) (1h)
The automatic titration system used in this study comprised
of an autoburette (Metrohm Dosimat 765 l mL syringe)
and Mettler Toledo MP230 pH meter with Metrohm pH
Yield: 79 %; IR (KBr) mmax 3400, 3035, 2970, 2926, 1670,
electrode (6.0133.100) and
a
reference electrode
1
and 1619 cm-1. H NMR (DMSO-d6) d 3.40 (m, 18H,
(6.0733.100). 0.1 M KCl electrolyte solution was used to
maintain the ionic strength. The temperature of the test
solutions was maintained in a thermostatic jacketed titra-
tion vessel at 25 0.1 °C using a Techne TE-8 J tem-
perature controller. The solution under investigation was
stirred vigorously during the experiment. A Gilson Mini-
plus#3 pump with speed capability (20 mL/min) was used
to circulate the test solution through a Hellem quartz flow
cuvette. For stability constant determinations, a 50 mm
path length cuvette was used, and for pKa determinations, a
cuvette path length of 10 mm was used. The flow cuvette
CH2), 3.71 (m, 12H, CH2), 4.19 (m, 6H, CH2), 7.16 (d, J =
7.5 Hz, 3H, Pyridinone C5-H), 8.05 (d, J = 7.5 Hz, 3H,
Pyridinone C6-H), 9.46 (br, 3H, NH); 13C NMR (DMSO-
d6) d 34.24 (CH2), 48.52 (CH2), 60.01 (CH2), 66.32 (CH2),
68.64 (CH2), 72.15 (CH2), 111.47 (C-5H in pyridinone),
135.21 (C-2 in pyridinone), 139.71 (C-6H in pyridinone),
143.38 (C-3 in pyridinone), 159.71 (C-4 in pyridinone),
163.53 (CO). ESI–MS m/z 822 ([M ? H]?). HRMS m/z
Calcd. for C36H52N7O15 [M ? H]? 822.3521, found
822.3543.
123

2356
Med Chem Res (2013) 22:2351–2359
MIC determination
was mounted on an HP 8453 UV–Visible spectrophotom-
eter. All instruments were interfaced to a computer and
controlled by a Visual Basic program. Automatic titration
and spectral scans adopted the following strategy: the pH
of a solution was increased by 0.1 pH unit by the addition
of KOH from the autoburette; when pH readings varied by
\0.001 pH unit over a 3 s period, an incubation period was
activated. For pKa determinations, a period of 1 min was
adopted; for stability constant determinations, a period of
5 min was adopted. At the end of the equilibrium period,
the spectrum of the solution was then recorded. The cycle
was repeated automatically until the defined end point pH
value was achieved. All the titration data were analyzed
with the pHab program (Gans et al., 1999). The species
plot was calculated with the HYSS program (Alderighi
et al., 1999). Analytical grade reagent materials were used
in the preparation of all solutions
All assays were cultured at 37 °C for 24 h in 15 9 75-mm
tubes. The incubation medium was BHI broth. All tubes
contained 80 lL of antimicrobial agent with a different
concentration ranging from 0 to 3,000 lg/mL, 20 lL of
bacterial inoculum, with a total volume of 100 lL. After
incubation at 37 °C for 2 h, 900 lL of sterilized water was
added to each tube to reach a final volume of 1 mL, and
cultured at 37 °C for 24 h. Minimum inhibitory concen-
trations (MIC) were determined by visual inspection of the
turbidity of broth in tubes (Barry, 1976). All assays were
carried out in triplicate.
Result and discussion
Chemistry
Considering that both iron(III) affinity and hydrophobicity
of iron chelators may affect their antimicrobial activity, we
have designed and synthesized a series of hexadentate 3-
hydroxypyridin-4-one derivatives (1a–1i) which have dif-
ferent structures to those of naturally occurring sidero-
phores, so as to investigate the structure-antimicrobial
activity relationship. The synthetic route of compound 1 is
outlined in Scheme 1. The benzylation of the starting
material, maltol (2) was achieved by the treatment with
benzyl chloride to obtain 3, which underwent the selective
oxidation of the methyl group at position-2 with selenium
dioxide to generate the aldehyde 4. Further oxidation of
aldehyde 4 with sodium hypochlorite and sulfanic acid
provided carboxylic acid 5 in good yield (Pace et al., 2004;
Puerta et al., 2006). Activation of carboxyl group of 5 was
achieved in the presence of N-hydroxysuccinmide (NHS)
and N,N’-dicyclohexylcarbodiimide (DCC) to give the
active ester 6, which was used in the next reaction without
isolation. Coupling of the active ester 6 with N,N-bis(2-
aminoethyl)ethane 1,2-diamine (TREN) gave 7, which was
reacted with different amines to provide a series of benzyl
protected hexadentate ligands 8. Finally, hydrogenation of
7 was carried out in the presence of Pd/C and hydrochloric
acid, providing the hydrochlorides of hexadentate 3-hy-
droxypyridin-4-ones 1 (a–i). All the hexadentate hydrox-
Antimicrobial assay
Bacterial strains
Pseudomonas aeruginosa, Staphylococcus aureus, and
Escherichia coli were purchased from CGMCC. Bacillus
subtilis and Bacillus cereus were separated from mussels
(see below). All bacteria were inoculated in a tube con-
taining an inclined plane of Brain–Heart Infusion (BHI)
agar and cultured at 37 °C for 24 h. This gel was then used
to inoculate into 5 mL of BHI broth and incubated at 37 °C
for 24 h before transfer 50 lL into another tube of fresh
BHI broth. This transfer was incubated at 37 °C to an
optical density of approximately 107 colony-forming units
(cfu/mL).
Isolation and identification of B. subtilis and B. cereus:
Mytilus edulis linne was obtained from a local fishing
company and was transported to the laboratory on ice.
Samples of 25 g muscle were homogenized in 250 mL of
0.1 % physiological peptone salt [PFZ 0.85 %NaCl (w/v)
and 0.1 % peptone (w/v)] for 60 s in a stomacher bag.
Suitable decimal dilutions were pour-plated on modified
plate count agar (PCA) for bacteria species. PCA agar
plates were incubated for 48 h at 30 °C. Representative
colonies were picked up randomly and purified by repeat-
edly streaking on appropriate agar medium. The isolates
were identified following the criteria outlined in Bergey’s
Manual of Systematic Bacteriology (Holt and Krieg, 1994).
Further characterization and confirmation was carried out
using a 6850 automated identification method (MIDI) and
PCR identification method.
1
ypyridinones have been characterized by H NMR, 13C
NMR, IR, ESI–MS, and HRMS. It is worthy to note that in
comparison with our reported synthesis of compound 1a,
which also starts from maltol (Piyamongkol et al., 2005),
the present synthetic method has the advantages of a
shorter synthetic procedure and applicability for the prep-
aration of a range of analog.
123

Med Chem Res (2013) 22:2351–2359
2357
Scheme 1 Reagents and
conditions: (a) CH3OH, NaOH,
BnCl. reflux 8 h; (b) PhBr,
SeO2, 159 °C, 12 h; (c)
CH3COCH3/H2O, NH2SO3H,
NaClO2, rt, 2 h; (d) THF, DCC,
NHS, rt, 3.5 h; (e) THF,
N(CH2CH2NH2)3, rt, overnight;
(f) CH3CH2OH, reflux, RNH2,
3 h; (g) CH3OH, Pd/C, H2.
5–6 h, rt
O
O
O
O
O
O
O
O
OBn
O
OBn
OH
OBn
d
OBn
O
a
c
b
N
COOH
O
CHO
O
O
O
2
4
5
6
3
O
O
N
O
O
OBn
NH
R
OBn
O
NH
f
e
R
N
O
O
N
N
O
O
N
N
H
H
BnO
BnO
HN
BnO
HN
BnO
O
O
O
O
O
N
O
R
7
8
O
N
OH
NH
R
O
1a: R = H
1b: R = Me
1c: R = Et
1d: R = n-Bu
1e: R = n-Hexyl
1f: R = CH2CH2OH
1g: R = CH2CH2OCH3
g
R
N
O
N
N
H
HO
HN
HO
O
O
O
1h: R = CH2CH2OCH2CH2OH
1i: R = CH2CH2NHCH2CH2OH
N
R
1
Physico-chemical characterization
determined using the same method as that for 1b, and in
similar fashion, all were found to possess six pKa values
(Table 1). For these four compounds, the pKa for the ter-
tiary amine function could not be detected using spectro-
photometric titration. However, we previously determined
the pKa values of 1a by potentiometric titration, and the
tertiary amine pKa value was found to be 4.25. We assume
that this value will be close to the pKa values of the cor-
responding amine functions in compounds (1b–1i).
In order to further demonstrate the high affinity for iro-
n(III), we selected some of the hexadentate chelators, and
evaluated their pKa values and stability constants for the
corresponding iron(III) complexes using an automated
titration system (Liu et al., 1999; Dobbin et al., 1993; Rai
et al., 1998), the titration data were analyzed with the pHab
(Gans et al., 1999).
pKa values
Iron(III) affinity
The pH dependence UV spectra of 1b (Fig. 2) was recor-
ded between 250 and 390 nm over the pH range 2–12 for
the free ligand. The speciation spectra demonstrate a clear
shift in kmax from 285 to 320 nm, which reflects the pH
dependence of the ligand ionization equilibrium. The
ligand group 1b can be considered as trimers of the cor-
responding bidentate ligands, and they, therefore, possess
two sets of intrinsic pKa values. Using the spectrophoto-
metric titration method, the pKa values of 1b obtained from
nonlinear least-squares regression analysis were found to
be 1.91, 2.56, 4.13, 6.92, 8.06, and 9.06. Of the six pKa
values, the three lower values correspond to the 4-oxo
function and the higher three correspond to the 3-hydroxyl
function. The pKa values of chelators 1c, 1g and 1h were
The stability constant of an iron-ligand complex is one of
the key parameters related to the chelation efficacy of a
ligand. The log stability constants of the series of chelators
1-iron(III) complexes were determined by spectrophoto-
metric titration against the hydroxyl anion. The logK1
values were found to be similar and all close to 32
(Table 1). As an example, a series of UV spectra of 1b in
the presence of iron at different pH values is shown in
Fig. 3. The pFe3? value, defined as the negative logarithm
of concentration of the free iron(III) in solution (when
[Fe3?
]
= 10-6M; [Ligand]total = 10-5M; pH = 7.4), is
total
a more suitable factor for comparison than the stability
constant, since this parameter takes into account the effect
of ligand basicity, denticity, and the degree of protonation.
123

2358
Med Chem Res (2013) 22:2351–2359
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.40
pH 11.892
pH 10..658
0.35
0.30
0.25
0.20
0.15
0.10
0.05
pH 2.141
290
pH 12.629
450
310
330
350
370
390
250
270
500
550
600
650
Wavelength (nm)
Wavelength (nm)
Fig. 2 UV spectra of 1b. [1b] = 222.6 lM (in 15.067 mL of 0.1 M
KCl), pH was changed from 2.141 to 11.892 by the addition of KOH
at 25 °C
Fig. 3 UV spectra of 1b with iron. [1b] = 32.7 lM, [Fe3?] =
29.7 lM (in 18.059 mL of 0.1 M KCl), pH was changed from 10.658
to pH 12.629 by the addition of KOH at 25 °C
Table 1 pKa values and iron affinities of hexadentate ligands
Antimicrobial evaluation
Ligands
pKa
logK1
pFe
The antimicrobial activity of these chelators was evaluated
using minimum inhibition concentration (MIC) assay. It
was found that all the iron chelators exhibited strong
inhibition against three Gram-positive bacteria (Staphylo-
ccocus aureus, Bacillus subtilis and Bacillus cereus) and
two Gram-negative bacteria (Escherichia coli and Pseu-
domonas aeruginosa) (Table 2). In the case of S. aureus,
the effect of 1a, 1b, 1c, 1e, and 1h are similar to that of
commercially available chelator diethylene-triamine pen-
taacetic acid (DTPA), with a MIC of 40 lg/mL, whereas
1d, 1f, and 1g have a MIC of 80 lg/mL. Against B. cereus
and B. subtilis, most of the chelators showed stronger
inhibitory activity than DTPA, with a MIC of 40 lg/mL. In
the case of E. coli and P. aeruginosa, almost all the che-
lators exhibited stronger inhibition than DTPA which has a
MIC of 80 lg/mL. 1g showed the strongest activity with a
MIC of 16 lg/mL. 1c exhibited the strongest overall
1a
1b
1c
1g
1h
0.66, 1.88, 3.6, 6.58, 7.65, 8.10
1.91, 2.56, 4.13, 6.92, 8.06, 9.06
1.91, 2.58, 4.23, 7.45, 8.26, 9.01
2.40, 3.76, 4.54, 7.30, 8.61, 9.27
1.89, 2.93, 4.31, 7.10, 7.75, 8.83
30.7
32.1
32.6
32.3
32.0
30.5
30.5
30.7
29.9
30.8
The pFe3? values of 1 (1b, 1c, 1g and 1h), calculated using
the measured stability constant and pKa values, are extre-
mely high, ranging between 29.9 and 30.8. These values
are close to that of 1a (pFe3? 30.5), suggesting that sub-
stituent in the 1-position of the pyridinone ring does not
appreciably influence the iron(III) affinity. This is probably
because of the group at 1-position has less effect on the
pKa value of 3-hydroxyl than the group at 2-position, and
the electron-withdrawing ability does not vary much
among the introduced substituents.
Table 2 Antimicrobial activity of chelators (MIC, lg/mL)
Compound
S. aureus
B. cereus
B. subtilis
E. coli
P. aeruginosa
Molecular weight
ClogP
DTPA
1a
40
40
40
40
80
40
80
80
40
80
40
40
40
80
40
40
80
40
80
40
40
40
80
40
40
40
40
80
80
24
24
40
80
40
16
40
80
24
24
16
40
40
40
16
40
393
557
599
641
725
809
689
731
821
-5.596
-4.505
-4.569
-3.638
-0.452
2.579
1b
1c
1d
1e
1f
-5.298
-4.595
-5.384
1g
1h
123

Med Chem Res (2013) 22:2351–2359
2359
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inhibitory activity when considering the entire group of
bacteria.
Most of the chelators investigated in this study were
found to possess stronger antimicrobial activity than
DTPA. This is presumably due to these chelators pos-
sessing a higher affinity for iron(III) than DTPA (logK =
28.6) (Sohnle et al., 2001). The calculated partition coef-
ficients (clogP) of the chelators (http://www.molinspir
ation.com/cgi-bin/properties), are also presented in
Table 2. These chelators are highly hydrophilic with the
exception of 1e (clogP 2.579). This hydrophilicity, together
with the relatively high molecular weights (Table 2) and
would suggest that these chelators and their iron complexes
will not be able to cross bilayer membranes by nonfacili-
tated diffusion. Thus, it is proposed that they inhibit the
growth of Gram-positive bacteria by scavenging iron in the
immediate environment around the bacteria and for Gram-
negative bacteria by entering and scavenging iron in the
periplasmic space. It is surprising that there is not a marked
difference between the antimicrobial properties of the
hydrophobic 1e (clogP 2.579) and the hydrophilic 1f (clogP
-5.298), and this further supports the concept of extra-
cellular iron chelation being responsible for the antimi-
crobial effect. Most of the chelators are more effective
against Gram-negative bacteria than Gram-positive bacte-
ria. This is probably due to the ready access to the peri-
plasmic space in Gram-negative bacteria.
Mitscher LA, Pillai SP, Gentry EJ, Shankel DM (1999) Multiple drug
resistance. Med Res Rev 19:477–496
Nikaido H, Zgurskaya HI (1999) Antibiotic efflux mechanisms. Curr
Opin Infect Dis 12:529–536
In conclusion, the inhibition of bacterial iron uptake
represents a promising alternative area of research for the
design of new antimicrobials, the hexadentate 3-hydroxy-
pyridin-4-one were found to possess strong inhibitory
activity against the growth of both Gram-positive and
Gram-negative bacteria. Overall, 1c showed optimal bac-
terial growth inhibitory effect. These hexadentate chelators
have potential as antimicrobial agents, particularly in the
treatment of external infections.
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characterisation of novel hexadentate 3-hydroxypyridin-4-one
ligands. Tetrahedron Lett 46:1333–1336
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KN, Cohen M (2006) Tris(pyrone) chelates of Gd(III) as high
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Lett 314:107–111
Acknowledgments This research work was financially supported
by the National Natural Science Foundation of China (No. 20972138),
Ningbo Science and Technology Bureau of China (No. 2007C10066),
Scientific Research Foundation for the Returned Overseas Chinese
Scholars, State Education Ministry of China ([2009]1590), and
Qianjiang Scholars Fund of Zhejiang Province (No. 2010R10051).
Rai BL, Dekhordi LS, Khodr HH, Jin Y, Liu ZD, Hider RC (1998)
Synthesis, physicochemical properties, and evaluation of N-
substituted-2-alkyl-3-hydroxy-4(1H)-pyridinones. J Med Chem
41:3347–3359
Sohnle PG, Hahn BL, Karmarkar R (2001) Effect of metals on
Candida albicans growth in the presence of chemical chelators
and human abscess fluid. J Lab Clin Med 137:284–289
Xu B, Kong XL, Zhou T, Qiu DH, Chen YL, Liu MS, Yang RH,
Hider RC (2011) Synthesis, iron(III)-binding affinity and in vitro
evaluation of 3-hydroxypyridin-4-one hexadentate ligands as
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