Design, synthesis, and antimicrobial evaluation of hexadentate hydroxypyridinones with high iron(iii) affinity

Article abstract of DOI:10.1111/cbdd.12358

A range of hexadentate 3-hydroxypyridin-4-ones (HPOs) with high affinity for iron(III) has been synthesized. The log stability constants of two HPO-iron complexes (logK1) were determined to be over 34, and pFe values of the two HPOs were determined to be over 31. Antimicrobial assay indicated that they are able to markedly inhibit the growth of both Gram-positive and Gram-negative bacteria. Compounds 14a and 14e were found to exhibit the strongest inhibitory activity against Staphyloccocus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli, with MIC values of 8, 8, 16, and 8 lg/mL, respectively. These results indicate that hexadentate 3-hydroxypyridin-4-ones have potential application as antimicrobial agents, especially in the treatment of wound infection.

Full text of DOI:10.1111/cbdd.12358

Received Date : 16-Feb-2014
Revised Date : 14-May-2014
Accepted Date : 17-May-2014
Article type
: Research Article
Design, synthesis and antimicrobial evaluation of hexadentate
hydroxypyridinones with high iron(III) affinity
Ming-Xia Zhanga, Chun-Feng Zhua, Ying-Jun Zhoua, Xiao-Le Kongb, Robert C Hiderb,
Tao Zhoua
*
a. School of Food Science and Biotechnology, Zhejiang Gongshang University,
Hangzhou, Zhejiang, 310018, P. R. China;
b Division of Pharmaceutical Science, King’s College London, Franklin-Wilkins
Building, 150 Stamford Street, London, SE1 9NH, UK.
Corresponding author: Tao Zhou
Zhejiang Gongshang University, Hangzhou, Zhejiang, 310018, P. R. China
Tel: (+86) 571 88071024-7587
Fax: (+86) 571 88905733;
E-mail: taozhou@zjgsu.edu.cn.
This article has been accepted for publication and undergone full peer review but has
not been through the copyediting, typesetting, pagination and proofreading process
which may lead to differences between this version and the Version of Record. Please
cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12358
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Running title: hexadentate hydroxypyridinones as antimicrobials
Abstract: A range of hexadentate 3-hydroxypyridin-4-ones (HPOs) with high affinity
for iron(III) have been synthesized. The log stability constants of two HPO-iron
complexes (logK1) were determined to be over 34, and pFe values of the two HPOs
were determined to be over 31. Antimicrobial assay indicated that they are able to
markedly inhibit the growth of both Gram-positive and Gram-negative bacteria.
Compounds 14a and 14e were found to exhibit the strongest inhibitory activity
against Staphyloccocus aureus, Bacillus subtilis, Pseudomonas aeruginosa and
Escherichia coli, with MIC values of 8, 8, 16 and 8 μg/mL, respectively. These results
indicate that hexadentate 3-hydroxypyridin-4-ones have potential application as
antimicrobial agents, especially in the treatment of wound infection.
Keywords: hydroxypyridinone; hexadentate; iron affinity; antimicrobial activity; iron
chelator
Introduction
Emergence of antibiotic-resistant bacteria and fungi is a growing global problem.1,2
There is an urgent need for the development of novel types of antimicrobial agents
with a wide antimicrobial spectrum. In order to grow and thrive, bacteria need iron
as a cofactor for many metabolic enzymes. Indeed, most microorganisms have
developed efficient methods of absorbing iron from the environment, many
involving the secretion of siderophores, which possess a high affinity and selectivity
for iron(III).3 For instance, Staphylococcus aureus has been demonstrated to use two
hydroxycarboxylate-type siderophores, staphyloferrin A and staphyloferrin B, via the
transporters Hts and Sir, respectively, to access the transferrin iron pool.4,5
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Pseudomonas aeruginosa produces two extensively characterized siderophores,
pyochelin and pyoverdin.6 In principle, the introduction of high affinity iron selective
chelating agents can limit the iron absorption of microorganism by competing with
siderophores for binding iron.7 However, the structure of antimicrobial chelators
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
transporter. In our previous work, hexadentate 3-hydroxypyridin-4-ones have been
demonstrated to inhibit the growth of both Gram-positive and Gram-negative
bacteria.8-10 However, some chelators with high iron-binding constants, for instance,
ethylenediamine-N, N’-bis(2-hydroxyphenyl) acetic acid (EDHPA, logK 34),
N,N’-bis(2-hydroxybenzyl) ethylenediamine-N,N’-diacetic acid (HEBD, logK 40), and
desferrioxamine (logK 30.4)11 have been reported to only possess a weak ability to
inhibit Gram-positive and Gram-negative bacterial growth,12 indicating that iron
affinity of the chelator is not the only factor which affects the antimicrobial potential
of an iron chelator. In this study, we explore the influence of hydrophobicity on the
antimicrobial efficacy of hexadentate 3-hydroxypyridin-4-ones.
Methods and Materials
Chemistry
All chemicals were of AR grade and used without any further purification. 1H NMR
and 13C NMR spectra were recorded on a Bruker Avance 500 spectrometer 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.
General procedure for preparation of 3
A mixture of 2 (10g, 46.3mmol), amine RNH2 (51 mmol), sodium hydroxide (4g,
100mmol) in methanol/water (40mL/40mL) was heated to reflux gently. The
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reaction was monitored by TLC. After completion of the reaction (about 2h), the
reactant was concentrated under reduced pressure to about half volume. Extracted
with dichloromethane (3×60mL), the combined organic layers were washed with
brine twice, dried over anhydrous sodium sulfate. After removal of the solvent, the
crude product 3 was obtained as a brown oil.
3-(Benzyloxy)-1,2-dimethylpyridin-4(1H)-one (3a)
Yield: 88%. 1HNMR (400MHz, CDCl3) δ 2.06 (s, 3H, CH3), 3.44 (s, 3H, CH3), 5.13
(s, 2H, CH2), 6.28 (d, J = 8.0Hz, 1H, Pyridinone C5-H), 7.18 (d, J = 8.0Hz, 1H,
Pyridinone C6-H), 7.23-7.39 (m, 5H, Ar). ESI-MS: m/z 230 ([M+H]+).
3-(Benzyloxy)-1-ethyl-2-methylpyridin-4(1H)-one (3b)
Yield: 86%. 1H NMR (400MHz, CDCl3) δ 1.28 (t, J = 7.6Hz, 3H, CH3), 2.08 (s, 3H,
CH3), 3.80 (d, J=7.6Hz, 2H, CH2), 5.18 (s, 2H, CH2), 6.44 (d, J=7.6Hz, Pyridinone C5-H),
7.20 (d, J=7.6Hz, Pyridinone C6-H), 7.26-7.38 (m, 5H, Ar). ESI-MS: m/z 244 ([M+H]+).
3-(Benzyloxy)-1-butyl-2-methylpyridin-4(1H)-one (3c)
Yield: 87%. 1H NMR (400MHz, CDCl3) δ 0.89 (t, J = 7.6Hz, 3H, CH3), 1.24-1.30 (m,
2H, CH2), 1.57-1.62(m, 2H, CH2), 2.07 (s, 3H, CH3), 3.71 (t, J = 7.6Hz, 2H, CH2), 5.21 (s,
2H, CH2), 6.41 (d, J = 7.6Hz, 1H, Pyridinone C5-H ), 7.16 (d, J = 7.6Hz, 1H, Pyridinone
C6-H), 7.25-7.39 (m, 5H, Ar). ESI-MS: m/z 272 ([M+H]+).
3-(Benzyloxy)-1-hexyl-2-methylpyridin-4(1H)-one (3d)
Yield: 90%. 1H NMR (400MHz, CDCl3) δ 0.90 (t, J = 7.6Hz, 3H, CH3), 1.21-1.30 (m,
6H, CH2), 1.58-1.62 (m, 2H, CH2), 2.07 (s, 3H, CH3), 3.75 (t, J = 7.6Hz, 2H, CH2), 5.23 (s,
2H, CH2), 6.43(d, J = 7.6Hz, 1H, Pyridinone C5-H), 7.17(d, J = 7.6Hz, 1H, Pyridinone
C6-H), 7.27-7.40 (m, 5H, Ar). ESI-MS: m/z 300 ([M+H]+).
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3-(Benzyloxy)-1-(2-hydroxyethyl)-2-methylpyridin-4(1H)-one (3e’)
Yield: 85%. 1H NMR (400MHz, CDCl3) δ2.11 (s, 3H, CH3), 3.84 (m, 4H, CH2), 4.95 (s,
2H, CH2), 6.15 (d, J = 6.0Hz, 1H, Pyridinone C5-H), 7.29-7.35 (m, 5H, Ar), 7.38(d, J=
6.0Hz, 1H, Pyridinone C6-H). ESI-MS: m/z 260 ([M+H]+).
Synthetic procedure for 3-(benzyloxy)-1-(2-(benzyloxy)ethyl)
-2-methylpyridin-4(1H)-one (3e)
To a solution of 3e’ (8g, 30.88mmol) in dry THF (80mL) was added sodium hydride
(1.12g, 46.6 mmol) and benzyl chloride (4.0g, 32 mmol). The mixture was refluxed
for 3h. Water was added dropwise cautiously to quench the reaction. The reactant
was concentrated and then was dissolved in dichloromethane, washed with brine
twice, dried over anhydrous sodium sulfate. After removal of the solvent, the crude
product 3e was obtained as a brown solid. Yield: 92 %. 1H NMR (400MHz, CDCl3) δ
2.06 (s, 3H, CH3), 3.56 (t, J = 4.0 Hz, 2H, CH2), 3.92 (t, J = 4.0 Hz, 2H, CH2), 4.40 (s, 2H,
CH2), 5.16 (s, 2H, CH2), 6.38 (d, J = 6.0 Hz, 1H, Pyridinone C5-H), 7.14 (d, J = 6.0 Hz,
1H, Pyridinone C6-H), 7.22-7.38 (m, 10H, Ph). ESI-MS: m/z 350 ([M+H]+).
3-(Benzyloxy)-1-(2-methoxyethyl)-2-methylpyridin-4(1H)-one (3f)
Yield: 84%. 1H NMR (400MHz, CDCl3) 2.09 (s, 3H, CH3), 3.16 (s, 3H, CH3), 3.48 (t,
J=4.0Hz, 2H, CH2), 3.80 (t, J = 4.0Hz, 2H, CH2), 5.15 (s, 2H, CH2), 6.35 (d, J =7.2Hz, 1H,
Pyridinone C5-H), 7.22-7.34 (m, 5H, Ar),7.37(d, J=7.2Hz, 1H, Pyridinone C6-H).
ESI-MS: m/z 274 ([M+H]+).
General procedure for preparation of 4
A mixture of 3 (10mmol), SeO2 (30mmol) in acetic acid/acetic anhydride
(25mL/25mL) was heated at 90-100oC for 6-8h. After removal of the solvent, the
residue was purified by silica gel column chromatography using ethyl
acetate/methanol (50:1～20:1) as an eluent to provide aldehyde 4 as a brown oil.
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3-(Benzyloxy)-1-methyl-4-oxo-1,4-dihydropyridine-2-carbaldehyde (4a)
Yield: 77%. 1H NMR (CDCl3, 400MHz) δ 3.78 (s, 3H, CH3), 5.50 (s, 2H, CH2), 7.30-7.36
(m, 5H, Ph), 6.49 (d, J = 7.6 Hz, 1H, C5-H in pyridinone), 7.16 (d, J =7.6 Hz, 1H, C6-H in
pyridinone), 10.05 (s, 1H, CHO). ESI-MS: m/z 244 ([M+H]+) .
3-(Benzyloxy)-1-ethyl-4-oxo-1,4-dihydropyridine-2-carbaldehyde (4b)
Yield: 79%. 1H NMR (CDCl3, 400MHz) δ 1.26 (t, J=7.2Hz, 3H, CH3), 4.19 (q, J=7.2Hz,
2H, CH2), 5.50 (s, 2H, CH2), 6.54 (d, J=7.6Hz, 1H, C5-H in pyridinone), 7.20 (d, J=7.6Hz,
1H, C6-H in pyridinone), 7.30-7.35 (m, 5H, Ph), 10.03 (s, 1H, CHO). ESI-MS: m/z 258
([M+H]+) .
3-(Benzyloxy)-1-butyl-4-oxo-1,4-dihydropyridine-2-carbaldehyde (4c)
Yield: 78%. 1H NMR (CDCl3, 400MHz) δ 0.88 (t, J=7.2Hz, 3H, CH3), 1.33 (m, 2H, CH2),
1.77 (m, 2H, CH2), 4.12 (t, J=7.2Hz, 2H, CH2), 5.48 (s, 2H, CH2), 6.50 (d, J=7.6Hz, 1H,
C5-H in pyridinone), 7.20 (d, J=7.6Hz, 1H, C6-H in pyridinone), 7.29-7.34 (m, 5H, Ar),
10.01 (s, 1H, CHO). ESI-MS: m/z 286 ([M+H]+).
3-(Benzyloxy)-1-hexyl-4-oxo-1,4-dihydropyridine-2-carbaldehyde (4d)
Yield: 79%. 1H NMR (CDCl3, 400MHz) δ 0.89 (t, J=7.2Hz, 3H, CH3), 1.33 (m, 6H, CH2),
1.77 (m, 2H, CH2), 4.13 (t, J=7.2Hz, 2H, CH2), 5.49 (s, 2H, CH2), 6.51 (d, J=7.6Hz, 1H,
C5-H in pyridinone), 7.19 (d, J=7.6Hz,1H, C6-H in pyridinone), 7.30 -7.34 (m, 5H, Ph),
10.01 (s, 1H, CHO). ESI-MS: m/z 314 ([M+H]+).
3-(Benzyloxy)-1-(2-(benzyloxy)ethyl)-4-oxo-1,4-dihydropyridine-2-carbaldehyde
(4e)
Yield: 71%. 1H NMR (CDCl3, 400MHz) δ 3.55 (t, J=4.4Hz, 2H, CH2), 4.34 (t, J=4.4Hz,
CH2), 4.37 (s, 2H, CH2), 5.48 (s, 2H, CH2), 6.51 (d, J=7.2Hz, 1H, C5-H in pyridinone),
7.14 (d, J=7.2Hz, 1H, C6-H in pyridinone), 7.25-7.33 (m, 10H, Ph), 9.95 (s, 1H, CHO).
ESI-MS: m/z 364 ([M+H]+).
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3-(Benzyloxy)-1-(2-methoxyethyl)-4-oxo-1,4-dihydropyridine-2-carbaldehyde (4f)
Yield: 80%. 1H NMR (CDCl3, 400MHz) δ 3.23 (s, 3H, CH3), 3.47 (t, J=4.4Hz, 2H, CH2),
4.31 (t, J=4.4Hz, 2H, CH2), 5.50 (s, 2H, CH2), 6.49 (d, J=7.6Hz, 1H, C5-H in pyridinone),
7.25 (d, J=7.6Hz, 1H, C6-H in pyridinone), 7.31-7.35 (m, 5H, Ph), 10.00 (s, 1H, CHO).
ESI-MS: m/z 288 ([M+H]+).
General procedure for preparation of 5
To a solution of 4（10mmol）in acetone/water (20mL/20mL) was added sodium
chlorite (12 mmol) and sulfamic acid (15 mmol). The mixture was stirred at room
temperature overnight. After filtration and washed with water and dried in a
desiccator, compounds 5 were obtained as white powders.
3-(Benzyloxy)-1-methyl-4-oxo-1,4-dihydropyridine-2-carboxylic acid (5a)
Yield: 65%. 1H NMR (DMSO, 400MHz) δ 3.60 (s, 3H, CH3), 5.03 (s, 2H, CH2), 6.31 (d,
J=7.2Hz, 1H, C5-H in pyridinone), 7.24-7.36 (m, 5H, Ar), 7.65 (d, J=7.2Hz, 1H, C6-H in
pyridinone). ESI-MS: m/z 260 ([M+H]+).
3-(Benzyloxy)-1-ethyl-4-oxo-1,4-dihydropyridine-2-carboxylic acid (5b)
Yield: 62%. 1H NMR (DMSO, 400MHz) δ 1.30 (t, J=7.6Hz, 3H, CH3), 4.31 (q, J=7.6Hz,
2H, CH2), 5.35 (s, 2H, CH2), 6.59 (d, J=7.2Hz, 1H, C5-H in pyridinone), 7.28 (d, J=7.2Hz,
1H, C6-H in pyridinone), 7.22-7.35 (m, 5H, Ph). ESI-MS: m/z 274 ([M+H]+).
3-(Benzyloxy)-1-butyl-4-oxo-1,4-dihydropyridine-2-carboxylic acid (5c) Yield:
55%. 1H NMR (DMSO, 400MHz) δ 0.86 (t, J=7.6Hz, 3H, CH3), 1.42 (m, 2H, CH2), 1.79
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(m, 2H, CH2), 4.25 (t, J=7.6Hz, 2H, CH2), 5.45 (s, 2H, CH2), 6.55 (d, J=7.2Hz, 1H, C5-H in
pyridinone), 7.20 (d, J=7.2Hz, 1H, C6-H in pyridinone), 7.25 -7.33 (m, 5H, Ph). ESI-
MS: m/z 302 ([M+H]+).
3-(Benzyloxy)-1-hexyl-4-oxo-1,4-dihydropyridine-2-carboxylic acid (5d)
Yield: 54%. 1HNMR (DMSO, 400MHz) δ 0.84 (t, J=6.8Hz, 3H, CH3), 1.20-1.26 (m,
6H, CH2), 1.66-1.72 (m, 2H, CH2), 3.88 (t, J=7.6Hz, 2H, CH2), 5.04 (s, 2H, CH2), 6.36 (d,
J=7.2Hz, 1H, C5-H in pyridinone), 7.27-7.42 (m, 5H, Ph), 7.72 (d, J=7.2Hz, 1H, C6-H in
pyridinone). ESI- MS: m/z 330 ([M+H]+).
3-(Benzyloxy)-1-(2-(benzyloxy)ethyl)-4-oxo-1,4-dihydropyridine-2-carboxylic acid
(5e)
Yield: 58%，1H NMR (DMSO, 400MHz) δ 3.69 (t, J=4.4Hz, 2H, CH2), 4.12 (s, 2H,
CH2) , 4.46 (t, J=4.4Hz, CH2), 5.06 (s, 2H, CH2), 6.33 (d, J=7.2Hz, 1H, C5-H in
pyridinone), 7.22-7.41(m, 10H, Ph), 7.69 (d, J=7.2Hz, 1H, C6-H in pyridinone). ESI-MS:
m/z 380 ([M+H]+).
3-(Benzyloxy)-1-(2-methoxyethyl)-4-oxo-1,4-dihydropyridine-2-carboxylic acid
(5f)
Yield: 60%，1H NMR (DMSO, 400MHz) δ 3.22 (s, 3H, CH3), 3.59 (t, J=5.2Hz, 2H,
CH2), 4.07 (t, J=5.2Hz, 2H, CH2), 5.04 (s, 2H, CH2), 6.34 (d, J=7.6Hz, 1H, C5-H in
pyridinone), 7.28-7.41 (m, 5H, Ph, 7.61 (d, J=7.6Hz, 1H, C6-H in pyridinone). ESI-MS:
m/z 304 ([M+H]+).
Synthetic procedure for 11a
Amine 10 was synthesized from nitromethane in four steps.13 To a solution of 10
(1.2g, 2.4 mmol) in CH2Cl2 (20 mL) cooled with an ice-bath was added triethyl amine
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(0.29 g, 2.88 mmol). Butyryl chloride (0.31 g, 2.88mmol) was then added dropwise.
The reaction mixture was stirred at 0oC for 3h, and then at room temperature for
additional 3h. The solution was washed with 5% NaHCO3 (2×20 mL) and brine, dried
over anhydrous Na2SO4, filtered and concentrated. The residue was purified by silica
gel column chromatography（cyclohexane : ethyl acetate, 1:1） to provide 11a as a
white solid. Yield: 85%. 1H NMR (CDCl3, 400MHz) δ 0.93 (t, J=7.2Hz, 3H, CH3),
1.30-1.38 (m, 2H, CH2), 1.46 (m, 27H, CH3), 1.47-1.53 (m, 6H, CH2), 1.58-1.66 (m, 6H,
CH2), 2.08 (t, J=7.2Hz, 2H, CH2), 3.08 (q, J=7.6Hz, 6H, CH2), 4.74 (s, 3H, NH), 5.21 (s,
1H, NH). ESI-MS: m/z 573 ([M+H]+).
Synthetic procedure for 11b
A mixture of N-carbobenzyloxy-L-leucine (0.265 g, 1 mmol), N-hydroxysuccinimide
(NHS) (0.134 g, 1.2 mmol) and N, N’-dicyclohexyl carbodiimide (DCC, 0.247 g,
1.2mmol) in DMF/CH2Cl2 (3mL/3mL) was stirred at room temperature overnight.
Compound 10 (0.502 g, 1mmol) was added to the reaction mixture and continued to
stir overnight.，After removal of the solvent, the residue was dissolved in CH2Cl2,
washed with saturated NaHCO3 and brine, dried over anhydrous Na2SO4, filtered and
concentrated. The residue was purified by silica gel column chromatography
(cyclohexane:EtOAc, 1:1) to provide 11b as a white solid. Yield: 88%. 1H NMR (DMSO,
400MHz) δ 0.87-0.96 (m, 6H, CH3), 1.47 (m, 27H, CH3), 1.49-1.58 (m, 7H, 3CH2 and
buried CH), 1.63 (m, 6H, CH2), 1.68 (m, 2H, CH2), 3.13 (q, J=7.6Hz, 6H, CH2), 3.98 (m,
1H, CH), 5.02 (s, 2H, CH2), 7.26 -7.35 (m, 5H, Ph). ESI-MS: m/z 751 ([M+H]+).
General procedure for preparation of 12.
A solution of 11 (1.5 g) in 96% formic acid (10 mL) was stirred at room temperature
for 24h. After removal of formic acid, toluene was added and then removed to get
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rid of residential formic acid. The crude product 12 was obtained as a pale yellow oil
in quantitative yield.
12a: Yield: 96%, 1H NMR (DMSO, 400MHz) δ 0.89 (s, J=7.6Hz, 3H, CH3), 1.50 (m, 2H,
CH2), 1.52 -1.64 (m, 12H, CH2), 2.06 (t, J=7.6Hz, 2H, CH2), 2.74 (t, J=8.0Hz, 6H, CH2).
ESI-MS: m/z 273 ([M+H]+).
12b: Yield: 97%. 1H NMR (DMSO, 400MHz) δ 0.85- 0.93 (m, 6H, CH3), 1.45-1.65 (m,
13H, CH2 and buried CH), 1.69 (m, 2H, CH2), 3.10 (t, J=8.0Hz, 6H, CH2), 5.01 (s, 2H,
CH2), 7.24 -7.33 (m, 5H, Ph). ESI-MS: m/z 451 ([M+H]+).
General procedure for preparation of 13
A mixture of 5 (5 mmol), NHS (0.67 g, 6 mmol) and DCC (1.236g, 6 mmol) in dry DMF
(20 mL) was stirred at room temperature overnight. 12 (1 mmol) was then added to
the reaction mixture, followed by the addition of triethylamine (3.6 mmol). The
resulting mixture was stirred at room temperature for 2 days. After removal of the
solvent, the residue was purified by column chromatography using MeOH/EtOAc
(1:8 to 1:3) as an eluent to give the desired product 13 as pale yellow solids.
13a. Yield: 62%. 1H NMR (DMSO-d6, 400MHz) δ 0.82 (t, J=7.2Hz, 3H, CH3), 1.32 (m,
2H, CH2), 1.35-1.49 (m, 6H, CH2), 1.51-1.61 (m, 6H, CH2), 1.98 (t, J=7.2Hz, 2H, CH2),
3.12 (q, J=6.4Hz, 6H, CH2), 3.51 (s, 9H, CH3), 5.03 (s, 6H, CH2), 6.19 (d, J=7.2Hz, 3H,
C5-H in pyridinone), 6.96 (br, 1H, NH), 7.23-7.34 (m, 15H, Ph), 7.57 (d, J=7.2Hz, 3H,
C6-H in pyridinone), 8.80 (t, J=5.6Hz, 3H, NH); 13C NMR (100Hz, DMSO) δ 173.17,
172.25, 161.17, 144.76, 140.54, 140.20, 138.19, 128.60, 128.29, 128.18, 117.48,
73.17, 57.51, 49.10, 40.94, 38.58, 32.68, 23.24, 19.36, 14.17. ESI-MS: m/z 996
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([M+H]+); HRMS: calcd. for C56H66N7O10 ([M+H]+) 996.4871, found 996.4848; calcd.
for C56H65N7O10Na ([M+Na]+) 1018.4691, found 1018.4675.
13b. Yield: 65%. 1H NMR (CD3OD, 400MHz) δ 0.92 (t, J=7.2Hz, 3H, CH3), 1.41 (t,
J=7.2Hz, 15H, 3CH3 and 3CH2), 1.55-1.60 (m, 8H, CH2), 2.08 (t, J=7.6Hz, 2H, CH2), 3.21
(t, J=7.2Hz, 6H, CH2), 3.98 (q, J=7.2Hz, 6H, CH2), 5.11 (s, 6H, CH2), 6.51 (d, J=7.2Hz,
3H, C5-H in pyridinone), 7.29-7.43 (m, 15H, Ph), 7.74 (d, J=7.2Hz, 3H, C6-H in
pyridinone); 13C NMR (100Hz, DMSO) δ173.11, 172.22, 161.16, 144.64, 139.92,
139.16, 138.27, 128.59, 128.21, 128.11, 118.01, 73.17, 57.50, 54.41, 49.07, 38.60,
32.84, 23.21, 19.41, 16.96, 14.19. ESI-MS: m/z 1038 ([M+H]+); HRMS: calcd. for
C59H72N7O10 ([M+H]+) 1038.5341, found 1038.5332; calcd. for C59H71N7O10Na
([M+Na]+) 1060.5160, found 1060.5148.
13c. Yield: 70%. 1H NMR (CDCl3, 400MHz) δ 0.81-0.89 (m, 12H, CH3), 1.25 -1.42 (m,
14H, CH2), 1.46-1.70 (m, 12H, CH2), 1.99 (t, J=7.6Hz, 2H, CH2), 3.12 (t, J=4.0Hz, 6H,
CH2), 3.79 (t, J=8.0Hz, 6H, CH2), 5.06 (s, 6H, CH2), 6.25 (d, J=7.2Hz, 3H, C5-H in
pyridinone), 7.28-7.39 (m, 15H, Ph), 7.68 (d, J=7.2Hz, 3H, C6-H in pyridinone); 13
NMR (100Hz, DMSO) δ173.11, 172.26, 161.06, 144.68, 139.94, 139.73, 138.22,
128.57, 128.21, 128.11, 117.64, 73.11, 57.54, 53.62, 49.13, 38.61, 33.10, 32.76,
23.26, 19.55, 19.38, 14.17, 13.92. ESI-MS: m/z 1122 ([M+H]+); HRMS: calcd. for
C65H84N7O10 ([M+H]+) 1122.6280, found 1122.6267; calcd. for C65H83N7O10Na
([M+Na]+) 1144.6099, found 1144.6107.
C
13d. Yield: 76%. 1H NMR (CDCl3, 400MHz) δ 0.89 (m, 12H, CH3), 1.08 (m, 6H, CH2),
1.29 (m, 24H, CH2), 1.53 (m, 2H, CH2), 1.77 (m, 6H, CH2), 1.92 (t, J=7.6Hz, 2H, CH2),
3.10 (t, J=7.6Hz, 6H, CH2), 3.75-3.79 (m, 6H, CH2), 4.98 (s, 6H, CH2), 6.21 (d, J=7.2Hz,
3H, C5-H in pyridinone), 7.15 (d, J=7.2Hz, 3H, C6-H in pyridinone), 7.25-7.37 (m, 15H,
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Ph); 13C NMR (100Hz, DMSO) δ173.11, 172.21, 161.07, 144.69, 139.91, 139.73,
138.25, 128.55, 128.20, 128.07, 117.63, 73.08, 57.44, 53.90, 49.08, 38.65, 32.76,
31.21, 31.02, 25.92, 23.28, 22.38, 19.38, 14.30, 14.17. ESI-MS: m/z 1206 ([M+H]+);
HRMS: calcd. for C71H95N7O10Na ([M+Na]+) 1228.7038, found 1228.7004.
13e. Yield: 68%，1H NMR (CD3OD, 400MHz) δ 0.88 (t, J=7.2Hz, 3H, CH3), 1.34 (m, 6H,
CH2), 1.52 (m, 8H, CH2), 2.04 (t, J=7.2Hz, 2H, CH2), 3.13 (t, J=7.6Hz, 6H, CH2), 3.72 (t,
J=4.8Hz, CH2), 4.11 (t, J=4.8Hz, 6H, CH2), 4.45 (s, 6H, CH2), 5.09 (s, 6H, CH2), 6.45 (d,
J=7.6Hz, 3H, C5-H in pyridinone), 7.25-7.38 (m, 30H, Ph), 7.68 (d, J=7.6Hz, 3H, C6-H in
pyridinone); 13C NMR (100Hz, DMSO) δ173.36, 172.27, 161.10, 144.75, 140.82,
139.77, 138.50, 138.21, 128.75, 128.58, 128.22, 128.12, 127.95, 127.74, 117.08,
73.17, 72.51, 69.10, 53.54, 49.10, 32.83, 29.53, 26.96, 23.25, 19.40, 14.18. ESI-MS:
m/z 1356 ([M+H]+); HRMS: calcd. for C80H89N7O13Na ([M+Na]+) 1378.6416, found
1378.6425.
13f. Yield: 71%，1H NMR (CD3OD, 400MHz) δ 0.92 (t, J=7.6Hz, 3H, CH3), 1.39 (m, 6H,
CH2), 1.57 (m, 8H, CH2), 2.09 (t, J=7.6Hz, 2H, CH2), 3.19 (t, J=7.2Hz, 6H, CH2), 3.31 (s,
9H, CH3), 3.65 (t, J=4.8Hz, CH2), 4.10 (t, J=4.8Hz, 6H, CH2), 5.12 (s, 6H, CH2), 6.47 (d,
J=7.2Hz, 3H, C5-H in pyridinone), 7.24-7.37 (m, 15H, Ph), 7.69 (d, J=7.2Hz, 3H, C6-H in
pyridinone); 13C NMR (100Hz, DMSO) δ173.27, 172.20, 161.05, 144.70, 140.67,
139.74, 138.21, 128.58, 128.22, 128.12, 117.12, 73.10, 71.16, 58.68, 57.44, 53.22,
49.08, 38.59, 32.80, 23.22, 19.37, 14.18. ESI-MS: m/z 1128 ([M+H]+); HRMS: calcd.
for C62H78N7O13 ([M+H]+) 1128.5658, found 1128.5674; calcd. for C62H77N7O13Na
([M+Na]+) 1150.5477, found 1150.5496.
13g. Yield: 74%，1H NMR (CD3OD, 400MHz) δ 0.90 (d, J=6.8Hz, 6H, CH3), 1.35-1.41
(m, 16H, 3CH3, 3CH2 and CH), 1.50-1.57 (m, 8H, CH2), 3.18 (t, J=7.6Hz, 6H, CH2), 3.97
(q, J=7.2Hz, 6H, CH2), 4.07-4.12 (m, 1H, CH), 5.01 (s, 2H, CH2), 5.11 (s, 6H, CH2), 6.50
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(d, J=7.2Hz, 3H, C5-H in pyridinone), 7.27-7.43 (m, 20H, Ph), 7.72 (d, J=7.2Hz, 3H,
C6-H in pyridinone). ESI-MS: m/z 1215 ([M+H]+); HRMS: calcd. for C69H83N8O12
([M+H]+) 1215.6130, found 1215.6086; calcd. for C69H82N8O12Na ([M+Na]+)
1237.5950, found 1237.5925.
13h. Yield: 72%，1H NMR (CDCl3, 400MHz) δ 0.89-0.97 (m, 15H, CH3), 1.30 -1.39 (m,
13H, 6CH2 and CH), 1.50-1.56 (m, 8H, CH2), 1.75-1.83 (m, 6H, CH2), 3.17 (t, J=8.0Hz,
6H, CH2), 3.93 (t, J=8.0Hz, 6H, CH2), 4.07-4.12 (m, 1H, CH), 5.01 (s, 2H, CH2), 5.12 (s,
6H, CH2), 6.50 (d, J=4.0Hz, 3H, C5-H in pyridinone), 7.28-7.43 (m, 20H, Ph), 7.72 (d,
J=4.0Hz, 3H, C6-H in pyridinone). ESI-MS: m/z 1299 ([M+H]+); HRMS: calcd. for
C75H95N8O12 ([M+H]+) 1299.7069, found 1299.7067; calcd. for C75H94N8O12Na
([M+Na]+) 1321.6889, found 1321.6903.
13i. Yield: 71%，1H NMR(CD3OD, 400MHz) δ 0.91 (d, J=6.8Hz, 6H, CH3), 1.32
-1.41 (m, 6H, CH2), 1.51-1.59 (m, 7H, 3CH2 and CH), 1.63-1.74 (m, 2H, CH2), 3.18 (t,
J=4.0Hz, 6H, CH2), 3.29 (s, 9H, CH3), 3.65 (t, J=4.0Hz, 6H, CH2), 4.03 (m, 1H, CH), 4.09
(t, J=4.0Hz, 6H, CH2), 5.02 (s, 2H, CH2), 5.12 (s, 6H, CH2), 6.45 (d, J=4.0Hz, 3H, C5-H in
pyridinone), 7.28-7.42 (m, 20H, Ar), 7.66 (d, J=4.0Hz, 3H, C6-H in pyridinone). ESI-MS:
m/z 1305 ([M+H]+); HRMS: calcd. for C72H89N8O15 ([M+H]+) 1305.6447, found
1305.6412; calcd. for C72H88N8O15Na ([M+Na]+) 1327.6267, found 1327.6260.
General procedure for preparation of 14
To a suspension of 13 (13a-i) (1mmol) and concentrated hydrochloric acid (1mL) in
MeOH (30 mL) was added 5% Pd/C (0.2 g). Hydrogenation was carried out at 30 psi
H2 for 8h. After filtration to remove the catalyst, the filtrate was concentrated to
dryness. The residue was purified by crystallization from methanol/acetone.
Hydrochlorides of hexadentate ligand 14 were obtained as white solids.
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14a. Yield: 95%，1H NMR (CD3OD, 500MHz) δ 0.97 (t, J=5.0Hz, 3H, CH3), 1.67 (m, 8H,
CH2), 1.88 (m, 6H, CH2), 2.25 (t, J=5.0Hz, 2H, CH2), 3.47 (t, J=7.5Hz, 6H, CH2), 4.10 (s,
9H, CH3), 7.27 (d, J=5.0Hz, 3H, C5-H in pyridinone), 8.25(d, J=5.0Hz, 3H, C6-H in
pyridinone). 13CNMR (100Hz, DMSO) δ 171.3, 160.6, 158.2, 141.9, 137.1, 136.1,
111.2, 57.0, 56.2, 43.2, 39.7, 37.4, 22.2, 18.8, 13.2. ESI-MS: m/z 726 ([M+H]+); HRMS:
calcd. for C35H48N7O10 ([M+H]+), 726.3462; found, 726.3496.
14b. Yield: 94%，1H NMR (CD3OD, 500MHz) δ 0.98 (t, J=7.5Hz, 3H, CH3), 1.57 (t,
J=7.0Hz, 9H, CH3), 1.67 (m, 8H, CH2), 1.88 (m, 6H, CH2), 2.22 (t, J=7.5Hz, 2H, CH2),
3.47 (t, J=7.5Hz, 6H, CH2), 4.41 (q, J=7.0Hz, 6H, CH2), 7.29 (d, J=6.5Hz, 3H, C5-H in
pyridinone), 8.33 (d, J=6.5Hz, 3H,C6-H in pyridinone). 13C NMR (100Hz, DMSO) δ
171.9, 161.3, 158.5, 142.7, 138.1, 136.7, 111.8, 57.1, 56.3, 40.1, 39.3, 37.9, 22.5,
18.8, 13.7, 13.3. ESI-MS: m/z 768 ([M+H]+); HRMS: calcd. for C38H54N7O10 ([M + H]+),
768.3931; found, 768.3944.
14c. Yield: 96%，1H NMR (CD3OD, 400MHz) δ 0.83-0.88 (m, 12H, CH3), 1.24 -1.49 (m,
14H, CH2), 1.46-1.77 (m, 12H, CH2), 2.04 (t, J=7.6Hz, 2H, CH2), 3.21-3.27 (m, 6H, CH2),
4.20 (t, J=6.4Hz, 6H, CH2), 7.42 (d, J=8.0Hz, 3H, C5-H in pyridinone), 8.31 (d, J=8.0Hz,
3H, C6-H in pyridinone). 13C NMR (100Hz, DMSO) δ 171.8, 161.4, 158.6, 142.8, 138.2,
136.8, 111.9, 57.0, 56.2, 40.0, 39.0, 37.9, 37.5, 32.3, 22.6, 18.8, 13.7, 13.3. ESI-MS:
m/z 852 ([M+H]+); HRMS: calcd. for C44H66N7O10 ([M+H]+), 852.4871; found,
852.4882.
14d. Yield: 96%，1H NMR (CD3OD, 400MHz) δ 0.90-0.99 (m, 12H, CH3), 1.25 -1.40 (m,
20H, CH2), 1.64 (m, 6H, CH2), 1.90 (m, 12H, CH2), 2.20 (t, J=7.6Hz, 2H, CH2), 3.44 (t,
J=6.4Hz, 6H, CH2), 4.33 (t, J=6.4Hz, 6H, CH2), 7.26 (d, J=6.4Hz, 3H, C5-H in
pyridinone), 8.29 (d, J=6.4Hz, 3H, C6-H in pyridinone). 13C NMR (DMSO, 100Hz) δ
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171.9, 161.3, 158.5, 142.7, 138.1, 136.7, 111.8, 57.0, 56.2, 40.1, 39.3, 37.9, 32.5,
30.4, 30.3, 25.1, 22.6, 21.7, 18.8, 13.6. ESI-MS: m/z 936 ([M+H]+); HRMS: calcd. for
C50H78N7O10 ([M+H]+), 936.5809; found, 936.5818.
14e. Yield: 93%，1H NMR (CD3OD, 400MHz) δ 0.98 (t, J=7.2Hz, 3H, CH3), 1.66 (m,
8H,CH2), 1.87 (m, 6H, CH2), 2.28 (t, J=7.2Hz, 2H, CH2), 3.45 (t, J=7.2Hz, 6H, CH2), 3.93
(t, J=4.8Hz, 6H, CH2), 4.55 (t, J=4.8Hz, 6H, CH2), 7.27 (d, J=6.8Hz, 3H, C5-H in
pyridinone), 8.24 (d, J=6.8Hz, 3H, C6-H in pyridinone). 13C NMR (100Hz, DMSO) δ
171.0, 161.3, 158.3, 142.7, 138.9, 136.5, 111.2, 68.7, 59.4, 56.4, 40.1, 37.4, 25.1,
22.2, 18.8, 13.3. ESI-MS: m/z 816 ([M+H]+) ); HRMS: calcd. for C38H54N7O13 ([M + H]+),
816.3779; found, 816.3798.
14f. Yield: 96%，1H NMR (CD3OD, 500MHz) δ 0.98 (t, J=7.5Hz, 3H, CH3), 1.66 (m, 8H,
CH2), 1.86 (m, 6H, CH2), 2.23 (t, J=7.5Hz, 2H, CH2), 3.42 (s, 9H, CH3), 3.46 (t, J=7.5Hz,
6H, CH2), 3.79 (t, J=5.0Hz, 6H, CH2), 4.55 (t, J=5.0Hz, 6H, CH2), 7.27 (d, J=7.0Hz, 3H,
C5-H in pyridinone), 8.23 (d, J=7.0Hz, 3H, C6-H in pyridinone). 13C NMR (100Hz,
DMSO) δ 171.0, 161.3, 158.3, 142.7, 138.9, 136.5, 111.2, 69.9, 58.8, 57.8, 56.2, 40.0,
39.6, 25.1, 22.6, 18.8, 13.6. ESI-MS: m/z 858 ([M+H]+); HRMS: calcd. for C41H60N7O13
([M+H]+), 858.4248; found, 858.4272.
14g. Yield: 94%，1H NMR (CD3OD, 400MHz) δ 0.86 (d, J=6.4Hz, 6H, CH3), 1.38 (t,
J=7.2Hz, 9H, CH3), 1.45-1.54 (m, 7H, 3CH2 and CH), 1.57-1.78 (m, 8H, CH2), 3.24 (m,
6H, CH2), 3.84 (m, 1H, CH), 4.20 (t, J=7.2Hz, 6H, CH2), 7.41 (d, J=7.2Hz, 3H, C5-H in
pyridinone), 8.32 (d, J=7.2Hz, 3H, C6-H in pyridinone). 13C NMR (100Hz, DMSO) δ
167.9, 161.1, 158.5, 142.3, 137.4, 136.6, 112.1, 58.2, 56.1, 51.8, 40.4, 40.0, 32.2,
32.1, 23.7, 22.4, 16.4, 13.2. ESI-MS: m/z 811 ([M+H]+); HRMS: calcd. for C40H59N8O10
([M+H]+), 811.4353; found, 811.4329.
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14h. Yield: 95%，1H NMR (CDCl3, 400MHz) δ 0.85-0.91 (m, 15H, CH3), 1.22 -1.29 (m,
6H, CH2), 1.42-1.81 (m, 21H, 10CH2 and CH), 3.24-3.28 (m, 6H, CH2), 3.82-3.87 (m,
1H, CH), 4.20 (t, J=7.2Hz, 6H, CH2), 7.41 (d, J=6.8Hz, 3H, C5-H in pyridinone),
8.28-8.30 (m, 3H, C6-H in pyridinone). 13C NMR (100Hz, DMSO) δ 168.41, 161.5,
158.8, 142.8, 138.0, 136.6, 111.9, 58.1, 56.2, 51.1, 40.5, 40.1, 32.4, 32.1, 23.6, 22.6,
22.3, 22.0, 18.9, 13.3. ESI-MS: m/z 895 ([M+H]+); HRMS: calcd. for C46H71N8O10
([M+H]+), 895.5292; found, 895.5310.
14i. Yield: 94%，1H NMR (CD3OD, 400MHz) δ 0.89 (t, J=6.4Hz, 6H, CH3), 1.39 -1.55
(m, 7H, CH2 and CH), 1.57-1.63 (m, 8H, CH2), 3.14-3.35 (m, 6H, CH2), 3.22 (s, 9H, CH3),
3.67 (t, J=4.8Hz, 6H, CH2), 3.81-3.87 (m, 1H, CH), 4.35 (t, J=4.8Hz, 6H, CH2), 7.31 (d,
J=6.8Hz, 3H, C5-H in pyridinone), 8.09 (d, J=6.8Hz, 3H, C6-H in pyridinone). 13C NMR
(100Hz, DMSO) δ 168.4, 161.5, 158.8, 142.8, 138.0, 136.6, 111.9, 70.0, 59.8, 58.0,
56.2, 51.1, 40.5, 40.1, 32.4, 32.5, 22.7, 18.8, 13.3. ESI-MS: m/z 901 ([M+H]+); HRMS:
calcd. for C43H65N8O13 ([M+H]+), 901.4670; found, 901.4643.
Physico-chemical properties of hexadentate hydroxypyridinones
pKa determination
The titration system used in this determination comprised an autoburette (Metrohm
Dosimat 765 l mL syringe) and a HP 8453 UV-visible spectrophotometer. 0.1M 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 7×10-5M. pKa values
were analyzed from these data by pHab.14
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Determination of iron(III) affinity
The automatic titration system used in this study comprised of an autoburette
(Metrohm Dosimat 765 liter ml syringe) and Mettler Toledo MP230 pH meter with
Metrohm pH electrode (6.0133.100) and a reference electrode (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 titration vessel at 25 oC
± 0.1 oC by using a Techne TE-8J temperature 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 was mounted on an HP 8453 UV-visible
spectrophotometer.15,16 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.14,16
The species plot was calculated with the HYSS program.17 Analytical grade reagent
materials were used in the preparation of all solutions.
Antimicrobial assay
Bacterial strains
P. aeruginosa, S. aureus, B. subtilis and E. coli were purchased from China General
Microbiological Culture Collection (CGMCC). The media used in this study were
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nutrient agar medium (NA) and tryptone soybean broth (TSB), which were purchased
from Beijing Land Bridge Technology Co. Ltd. These four bacterial strains were
inoculated in a tube containing an inclined plane of NA and cultured at 37 oC for 24
h. This gel was then used to inoculate into 5 mL of TSB and incubated at 37 oC for 24
h before transfer 50 μL into another tube of fresh TSB. This transfer was incubated at
37 °C to an optical density of approximately 5.0×105 colony-forming units (CFU/ml).
Determination of Minimum Inhibitory Concentration (MIC)
All assays were cultured at 37 oC for 24 h in 10×100-mm tubes. The incubation
medium was Trypticase Soy Broth (TSB). All tubes contained 80 µL of antimicrobial
agent with different concentrations up to 2048 µL /mL, 20µL of bacterial inoculum
with 1-2×105 cells/mL , with a total volume of 100µL. After incubation at 37℃ for 2
h, 900 µL of sterilized TSB was added to each tube to reach a final volume of 1 mL ,
and cultured at 37℃ for 24 h. Minimum inhibitory concentrations (MIC) were
determined by visual inspection of the turbidity of broth in tubes.18 All assays were
carried out in triplicate.
Results and Discussion
Chemistry
Hexdentate ligands can be formed by conjugating three bidentate ligands onto
a suitable tripodel backbone. However, in order for the ligand to adopt the correct
geometry for iron(III) binding, it is essential that the backbone be connected to the
ring at the ortho position relative to the oxygen anion,19 namely, connecting to
position-2 or 5 at pyridinone ring. In this work, we firstly designed and synthesized a
range of benzyl protected bidentate hydroxypyridinones (HPO) containing free
carboxyl group at position-2 starting from maltol (Scheme 1). The benzylation of the
starting material, maltol (1) was achieved by the treatment with benzyl chloride to
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obtain 2. Treatment of 2 with primary amine under basic conditions with reflux
provided HPO derivatives 3 in 84-90% yield, which underwent the selective oxidation
of the methyl group at position-2 with selenium dioxide in acetyl anhydride to
generate aldehydes 4. Further oxidation of 4 in the presence of sodium hypochlorite
and sulfanic acid provided the carboxylic acids 5 in good yield. The tripodal
molecules (12) containing three free amino groups were synthesized starting from
nitromethane (6) (Scheme 2). In the presence of Triton B, nitromethane underwent a
Michael addition with acrylonitrile to produce 7 in good yield. Reduction of 7 with
B2H6 generated 8, in which three amino groups were then protected with Boc to give
9. The nitro group in 9 was reduced by hydrogenation in the presence of Raney
nickel to produce 10.13 Reaction of 10 with butyryl chloride gave 11a; coupling of 10
with N-Cbz-L-leucine in the presence of
O-(6-chloro-1-hydrocibenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate (HCTU) provided 11b. Treatment of 11a and 11b with formic
acid generated triamines 12a and 12b, respectively. Activation of 12 to an active
ester was achieved in the presence of N-hydroxysuccinimide (NHS) and (DCC).
Hexadentate hydroxypyridinones 14 were synthesised by the coupling of the active
ester with triamines followed by the removal of the benzyl protecting groups under
hydrogenation (Scheme 3).
Physico-chemical characterisation
pKa values
The pH dependent UV spectra of 14c (Figure 1) was recorded between 250 and 400
nm over the pH range 1.728 - 11.754 for the free ligand. The speciation spectra
demonstrate a clear shift in λmax from 290 to 320 nm, which reflects the pH
dependence of the ligand ionization equilibrium. The ligand 14 can be considered as
a trimer of the corresponding bidentate ligand and it therefore possesses two sets of
intrinsic pKa values. Using the spectrophotometric titration method, the pKa values
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of 14c obtained from nonlinear least-squares regression analysis were found to be
1.99, 2.29, 2.42, 7.46, 8.37 and 8.95. Of the six pKa values, the three lower values
correspond to the 4-oxo functions and the higher three correspond to the 3-hydroxyl
functions. The pKa values of chelator 14h were determined to be 1.92, 2.72, 3.95,
7.43, 8.40, 9.28 and 10.79 using the same method as that for 14c. The value 10.79 is
assigned to the amino group of leucine residue.
Iron(III) affinity
The stability constant of an iron-ligand complex is one of the key parameters
related to the chelation efficacy of a ligand. The UV spectra of 14c in the presence of
iron at different pH values are shown in Figure 2. The stability constant of the
iron(III) complexes (logK1) of 14c and 14h were determined to be 34.9 and 34.6,
respectively.
The pFe3+ value, defined as the negative logarithm of concentration of the free
iron(III) in solution (when [Fe3+]Total = 10-6M; [Ligand]Total = 10-5M; pH = 7.4), is a more
suitable parameter for comparison than the stability constant, since it takes into
account the effect of ligand basicity, denticity and the degree of protonation. The
pFe3+ values of 14c and 14h, calculated by using the measured stability constant
logK1 and pKa values, are extremely high, being 31.9 and 31.3 respectively. For
comparative purposes, the pFe3+ value for most widely clinically used iron chelator
desferioxamine is 26.6.
Antimicrobial evaluation
The antimicrobial activity of these hexadentate hydroxypyridinones was evaluated
using minimum inhibition concentration assay. A clinically used bidentate
hydroxypyridinone (Deferiprone) and a commercially available chelator,
ethylenediamine tetraacetic acid (EDTA), were adopted as comparators. It was found
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that all the iron chelators exhibited marked inhibition against tested Gram-positive
bacteria (S. aureus and B. subtilis) and two Gram-negative bacteria (E. coli and P.
aeruginosa) (Table 1). Deferiprone was found to possess the weakest inhibition
against all of the four tested bacterial strains, which can be attributed to its relatively
lower iron affinity (pFe = 20.5). In the case of S. aureus, 14a, 14b, 14c, 14e, 14f and
14h were found to exhibit strong inhibitory activity, with a MIC of 8 μg/mL, which is
lower than EDTA (MIC 32 μg/mL), whereas 14g has a MIC of 512 μg/mL. Against B.
subtilis, most of the chelators (14a, 14b, 14c, 14e, 14f and 14h) showed stronger
inhibitory activity than EDTA, with a MIC of 8 μg/mL, while 14g and 14i showed
relatively low inhibitory effect. In the case of P. aeruginosa, almost all the chelators
exhibited stronger inhibition (MIC 16-32 μg/mL) than EDTA (MIC 128μg/mL), with
the exception of 14i, which has a similar effect to EDTA. In the case of E. coli, again
almost all the chelators were found to possess stronger inhibition (MIC 8-32 μg/mL)
than EDTA (MIC 64 μg/mL), with the exception of 14i, which has a similar effect to
EDTA.
Most of the chelators investigated in this study were found to possess stronger
antimicrobial activity than that of EDTA. This is presumably due to these chelators
possessing a higher affinity for iron(III) than EDTA (logK = 28.6).20 Based on the
similar affinity constants for iron(III) of compounds 14c and 14h, it is most probable
that the affinity for iron(III) remains largely unchanged for the entire group (14a-14i)
and thus any variation of antibacterial influence is unlikely to be related to
differences in iron affinity. The calculated partition coefficients (ClogP) of the
chelators,21 are also presented in Table 1. Overall, 14i and 14g showed relatively
weaker inhibitory effect than the other hexadentate hydroxypyridinones, indicating
that the presence of a charged amino group has a detrimental influence on
antibacterial effect – possibly by adversely influencing the penetration of
compounds into the surface of bacteria. Significantly compound 14h is not so
severely influenced by the presence of the amino function and this compound is
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markedly more hydrophobic than both 14i and 14g. On consideration of the ClogP
values of the noncharged chelators (14a-14f), those with values falling in the range
-4.38 to +1.74 all have similar efficacies. Whereas the most hydrophobic chelator
14d with a ClogP value of +4.77 has a low efficacy against most of the organisms.
Thus in summary it would appear that the non-charged state of chelator is important
for activity as is a reasonable water solubility (negative ClogP value). It is proposed
that these chelators inhibit the growth of Gram-positive bacteria by scavenging iron
in the immediate environment on the surface of the bacteria and for Gram negative
bacteria by entering and scavenging iron in the periplasmic space.
Conclusion
The inhibition of bacterial iron uptake represents a promising alternative approach
for the design of new antimicrobials, the hexadentate 3-hydroxypyridin-4-ones were
found to possess strong inhibitory activity against the growth of both Gram-positive
and Gram-negative bacteria. Overall, the more hydrophilic 14a and 14e showed
optimal bacterial growth inhibitory effect. These hexadentate chelators have
potential as antimicrobial agents, particularly in the treatment of external infections.
Acknowledgments
The financial support from the National Natural Science Foundation of China (No.
20972138), and Qianjiang Scholars Fund of Zhejiang Province (No. 2010R10051) is
gratefully acknowledged.
Disclosure
All authors declare that they have no conflict of interest.
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21. http://www.molinspiration.com/cgi-bin/properties
Scheme 1
O
O
O
O
OBn
OBn
OBn
OH
a
b
c
N
R
N
R
CHO
O
O
2
1
3
4
O
OBn
d
5a: R=CH3
5b: R=CH3CH2
5c: R=n-C4H9
5d: R=n-C6H13
5e: R=CH3CH2OBn
5f: R=CH2CH2OCH3
N
R
COOH
5
Scheme 1
Reagents and conditions：(a) BnCl, NaOH, CH3OH, reflux, 7-8h; (b) RNH2, NaOH,
CH3OH/H2O, reflux, 2.5h; (c) SeO2, CH3COOH/(CH3CO)2O, reflux; (d) NH2SO3H,NaClO2
，CH3COCH3/H2O
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Scheme 2
NHBoc
NH2
CN
CN
c
b
NHBoc
NH2
O2N
CN
O2N
CH3NO2
O2N
a
NHBoc
NH2
CN
7
6
8
9
NH2
NHBoc
NHBoc
O
O
e
f
d
NH2
R
N
H
NHBoc
NHBoc
NHBoc
H2N
R
N
H
NH2
NHBoc
12
11
10
R = n-C3H7 (a),
(b)
NHCbz
Scheme 2
Reagents and conditions：(a) Triton B, 30 oC, overnight.(b) BH3-THF, 24h; (c) (Boc)2O,
CH3OH; (d) H2, Raney-Ni, EtOH, 5d; (e) A. for 11a: n-C3H7COCl, Et3N,DCM; B. for 11b:
Cbz-L-Leu, NHS, DCC, Et3N, DMF. (f) HCOOH, rt, 24h.
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Scheme 3
O
BnO
O
H
N
NH2
O
N
R
O
O
OBn
O
a
BnO
H
+
NH2
N
R1
N
H
R1
N
H
N
R
COOH
N
R
O
NH2
NH
OBn
12
O
O
13
5
N
R
O
HO
O
H
N
N
R
O
R1 = n-C3H7
O
R1 =
HO
b
H
NH2
N
R1
N
14a: R=CH3
H
N
R
OH
O
14g: R=CH2CH3
14h: R=n-C4H9
14i: R=CH2CH2OCH3
14b: R=CH2CH3
14c: R=n-C4H9
14d: R=n-C6H13
NH
O
O
14e: R=CH2CH2OH
14f: R=CH2CH2OCH3
14
N
R
Scheme 3
Reagents and conditions：(a) NHS,DCC,DMF,4d. (b) CH3OH, Pd/C, 8h.
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Figure Legends
Figure 1. Titration of 14c. [14c] = 67.8 μM, pH was changed from 1.728 to 11.754
by the addition of KOH in 20.290 mL of 0.1M KCl at 25 oC.
Figure 2. Titration of 14c with iron(III). [14c]= 23.3 μM, [Fe3+]= 22.5 μM with the
addition of KOH, start in 20.058 ml of 0.1M KCl at 25oC, pH from 10.584 to pH
12.980.
Figure 1
1.70
1.50
1.30
1.10
pH 11.754
0.90
0.70
0.50
0.30
0.10
pH 1.728
290
370
250
270
310
33
350
390
Wavelength (nm)
Figure 2
0
0.45
pH 10.584
0
0.35
0
0.25
0
0.15
0
0.05
pH 12.980
460
620
500
Wavelength (nm5)80
540
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Table 1. Antimicrobial activity of chelators (MIC, μg/mL)
Compounds
Deferiprone
EDTA
14a
S. aureus
B. subtilis
P. aeruginosa
E. coli
512
64
8
ClogP
-0.60
-5.19
-2.57
-1.44
1.74
512
32
8
512
64
8
512
128
16
14b
8
8
32
8
14c
8
8
32
8
14d
32
8
64
8
32
32
8
4.77
14e
16
-4.38
-2.62
-4.87
0.82
14f
8
8
32
16
32
8
14g
512
8
256
8
32
14h
32
14i
64
512
128
64
-3.54
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