Synthesis, iron(III)-binding affinity and in vitro evaluation of 3-hydroxypyridin-4-one hexadentate ligands as potential antimicrobial agents

Article abstract of DOI:10.1016/j.bmcl.2011.08.097

Iron is a critical element for the survival of bacteria. We have designed and synthesized two novel 3-hydroxypyridin-4-one hexadentate ligands with high affinity for iron(III), which disrupt bacterial iron absorption. Biological studies demonstrate that these two chelators have significant inhibitory effect against both Gram-positive and Gram-negative bacteria, and therefore have potential as antimicrobial agents.

Full text of DOI:10.1016/j.bmcl.2011.08.097

Bioorganic & Medicinal Chemistry Letters 21 (2011) 6376–6380
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis, iron(III)-binding affinity and in vitro evaluation of
3-hydroxypyridin-4-one hexadentate ligands as potential antimicrobial agents
Bo Xu a, Xiao-Le Kong b, Tao Zhou a, , Di-Hong Qiu c, Yu-Lin Chen b, Mu-Song Liu d, Rong-Hua Yang a,
⇑
Robert C. Hider b
a School of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou, Zhejiang 310012, PR China
b Division of Pharmaceutical Science, King’s College London, Franklin–Wilkins Building, 150 Stamford Street, London SE1 9NH, UK
c Faculty of Life Science and Biotechnology, Ningbo University, Ningbo, Zhejiang 315211, PR China
d College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou 310014, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2011
Revised 17 August 2011
Accepted 24 August 2011
Available online 28 August 2011
Iron is a critical element for the survival of bacteria. We have designed and synthesized two novel
3-hydroxypyridin-4-one hexadentate ligands with high affinity for iron(III), which disrupt bacterial iron
absorption. Biological studies demonstrate that these two chelators have significant inhibitory effect
against both Gram-positive and Gram-negative bacteria, and therefore have potential as antimicrobial
agents.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Iron chelator
3-Hydroxypyridin-4-one
Hexadentate
Antimicrobial activity
As metal ions are essential for all living processes, using selec-
tive metal chelators as therapeutical agents, namely chelation
therapy, has received increasing attention. Iron chelators, for in-
stance desferrioxamine B (DFO),1 deferiprone2 and exjade3 have
been used for the treatment of iron overload disorders associated
with b-thalassaemia and sickle cell anaemia. Iron is an essential
element for the growth of virtually all bacteria and fungi, conse-
quently microorganisms have developed efficient methods of
absorbing iron from the environment. Many microorganisms se-
crete siderophores in order to scavenge iron.4 Such methods of up-
take can be circumvented by the introduction of high affinity iron
selective chelating agents. The iron affinity of these agents must be
extraordinally high, so that they can efficiently compete with sid-
erophores. Furthermore, the structure of chelators should differ
appreciably from those of siderophores, otherwise the iron-chela-
tor complex will be recognized by the iron-siderophore receptors
and utilized by the microorganism. Antimicrobial activity of chela-
tors has been previous reported,5–9 however, most chelators inves-
tigated for this purpose have been bidentate ligands, which possess
a relatively low affinity for iron(III).10 In contrast hydroxypyridi-
none hexadentate ligands, possess a high affinity for iron(III)11,12
and have different structures and net charges to those of typical
siderophores. Thus, in principle, they should inhibit the growth
of bacteria. Indeed, our recent work has demonstrated that the
hexadentate hydroxypyridinone (1) exhibits a stronger inhibitory
effect on the growth of bacteria than the commercially available
chelator DTPA.13 Herein we report the synthesis, iron-chelating
properties and antimicrobial activity of two structurally related
3-hydroxypyridin-4-one hexadentate ligands.
The synthesis of the two 3-hydroxypyridin-4-one hexadentate
ligands, begins with commercially available kojic acid (2) and is
presented in Scheme 1. The benzyl-protected bidentate hydrox-
ypyridinone, which contains
a free amino group, 2-(amino-
methyl)-3-(benzyloxy)-1,6-dimethylpyridin-4(1H)-one (6) was
synthesized from kojic acid by following an established procedure
with slight modifications.14 Kojic acid was treated with thionyl
chloride, followed by reduction with zinc in hydrochloric acid to
give allomaltol (3). The a-position with respect to the ring hydro-
xyl was then functionalized in an analogous fashion to that of the
aldol condensation, followed by benzyl protection of the 3-hydro-
xyl group, giving compound 4. The 2-hydroxyl function of 4 was
protected using 3,4-dihydro-2H-pyran before reaction with methy-
lamine. The pyran protecting group was subsequently removed un-
der mild acid conditions, yielding product 5. Amine 6 was obtained
by the treatment of 5 with thionyl chloride, followed by the reac-
tion with ammonium solution. Amine 6 was conjugated to the two
triacids, nitrilotriacetic acid and 3,30,300-nitrilotripropionic acid, via
amide bonds in the presence of 1-hydroxybenzotriazole (HOBt)
and 1,3-dicyclohexylcarbodiimide (DCC) in N,N-dimethylformam-
ide (DMF) at room temperature, providing the protected hexaden-
tate ligands 7.15 Deprotection of the benzyl groups on 7 was
⇑
Corresponding author. Tel.: +86 57188071024/7587; fax: +86 571 88905733.
E-mail address: taozhou@zjgsu.edu.cn (T. Zhou).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.08.097

B. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6376–6380
6377
achieved by hydrogenation in the presence of palladium/charcoal,
generating the hexadentate chelators 8.16 All the compounds have
0.8
0.6
0.4
0.2
0.0
pH 11.2
been fully characterized by 1H NMR, 13C NMR, MS and HRMS.
O
N
HO
HN
H
O
H
N
O
N
N
H
pH 1.93
OH
NH OH
O
O
O
N
H
250
290
Wavelength (nm)
330
270
310
350
1
Figure 1. UV spectra of 8a. [8a] = 24.5 lM, pH was changed from 1.93 to 11.20 by
the addition of KOH.
O
N
NH
O
H
N
The carbon chain of the tripodal scaffold in the hexadentate
chelator 8a is shorter than that of 8b. Thus, although the hexaden-
tate chelators 8a and 8b possess the same chelating group, the sta-
bility of their iron complexes is predicted to be slightly different.
In order to further demonstrate the ligand affinity, both pKa val-
ues of the hexadentate chelators 8a and 8b and their stability con-
stants for the corresponding iron(III) complexes were evaluated
using an automated titration system.17–19 All the titration data
were analyzed using the pHab software.20 The pH dependence
UV spectra of 8a (Fig. 1) was recorded between 250 and 350 nm
over the pH range 1.93–11.20 for the free ligand. The speciation
spectra demonstrate a clear shift in kmax from 280 to 310 nm,
which reflects the pH dependence of the ligand ionization equilib-
rium. The pKa values of 8a obtained from nonlinear least-squares
regression analysis are 2.58, 3.30, 3.97, 8.41, 9.30 and 10.12. Of
the six pKa values, the lower three values correspond to the
4-oxo functions and the higher three correspond to the 3-hydroxyl
functions. Surprisingly, the pKa for the tertiary amine function of
O
HN
9
O
OH
N
10
O
O
O
O
O
O
OBn
OBn
OBn
OH
OH
OH
c
d
b
a
NH2
OH
N
HO
N
O
O
2
3
4
6
5
O
O
HO
BnO
N
N
NH
NH
f
O
O
e
n
n
O
O
N
N
n
n
OH
OBn
OH
n
N
OBn
HN
n
N
O
HN
O
H
H
O
O
O
O
N
N
N
N
7a: n = 1
7b: n = 2
8a: n = 1
8b: n = 2
Scheme 1. Reagents and conditions: (a) (i) SOCl2, (ii) Zn/HCl, 70%; (b) (i) HCHO, (ii) BnCl, 65%; (c) (i) 2,3-dihydro-4H-pyran, TsOH, (ii) MeNH2, (iii) 6 M HCl, 85%; (d) (i) SOCl2,
(ii) 37% NH3, MeOH, 72% in two steps; (e) triacid, HOBt, DCC, DMF, rt, 88% for 7a and 83% for 7b; (f) H2 (30 psi), 5% Pd/C (20% W/W), MeOH, HCl, 93% for 8a and 90% for 8b.

6378
B. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6376–6380
A 12
B
10
8
6
4
2
0
12
10
8
6
4
2
0
0
0.5
1
1.5
2
2.5
3
0
0.5
1
1.5
2
2.5
3
KOH (mL)
KOH (mL)
Figure 2. A: Potentiometric titration of 8a (18.5 mg in 20 mL KCl) at 25 °C. B: Potentiometric titration of 8b (18.9 mg in 20 mL KCl) at 25 °C.
pH 12 while 8a starts at pH 12.5. In order to comprehend the supe-
rior iron(III) affinity of 8a, we applied molecular dynamics simula-
tion studies on these two iron complexes.22 After 48,000
simulations, an optimized structure was obtained for each iron(III)
complex model (Fig. 5). The corresponding bidentate iron(III) com-
plex with 1,2-dimethyl-3-hydroxypyridin-4-one (deferiprone, 10)
was also investigated using the same process. We found that 8a
has less octahedron distortion than 8b, when compared with the
bidentate counterpart (Table 1). For bidentate ligands there is no
0.6
0.5
0.4
0.3
0.2
0.1
0.0
A
0.6
pH 3.4
0.5
230
240
250
260
270
280
290
300
0.4
0.3
0.2
Wavelength (nm)
Figure 3. Spectrophotometric titration of 9 (140.6
25 °C, pH was changed from7.17 to 0.192.
lM in 20.006 mL 0.1 M KCl) at
8a was not detected, even with potentiometric titration (Fig. 2A).
The corresponding pKa values of 8b were determined as: 2.98,
3.59, 3.89, 8.03, 8.94 and 9.81. Again, the tertiary amine pKa value
was not detected during the spectrophotometric titration although
it was clearly identified by potentiometric titration (Fig. 2B) where
a buffer range with a corresponding pKa value (6.18+/ꢀ0.072) was
observed. In order to investigate further the pKa value associated
with the tertiary amine function of compound 8a, an analogue
based on 8a which has the 3-hydroxypyridin-4-one groups re-
placed by benzene (9) was also synthesized using a reported meth-
od.21 Only one pKa value exists for this compound and by
spectrophotometric titration a surprisingly low pKa value (1.41+/
ꢀ0.05) was identified (Fig. 3). This indicates that the three amide
functions adjacent to the tertiary amine function, exert a strong
electron withdrawing effect and considerably decrease the charge
density on the central nitrogen atom. In contrast with 8b, due to
the longer carbon chains there is a much weaker electron with-
drawing effect. Significantly the pKa value of 1 is intermediate be-
tween the two values obtained for 8a and 8b, namely 4.52.11
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 8a and 8b for iron(III) were determined
to be 35.07 and 33.76, respectively, using a spectrophotometric
titration against the hydroxyl anion (Fig. 4A). Under exactly the
same experimental conditions, the 8a-iron(III) complex was found
to be stable at higher pH values than that of the 8b-iron(III) com-
plex. As depicted in Figure 4B (455 nm) 8b starts to dissociate at
pH 13.0
0.1
0.0
440
520
600
480
560
Wavelength (nm)
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
B
11.5
12
12.5
13
pH
Figure 4. A: UV spectra of 8a in the presence of iron(III). [8a] = 32.7
M. The pH was changed from 3.436 to 13.016 by the addition of
KOH. B: The stability of the iron complexes of 8a (s) and 8b (d) as a function of pH,
lM,
[Fe3+] = 29.8
l
as monitored by the absorption at 455 nm.

B. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6376–6380
6379
Figure 5. A: Optimized structures of 8a-iron complex (left) and 8b-iron complex (right) obtained by molecular dynamic simulation studies.21 C(green); O(red); iron(yellow)
N(blue) H(white).
Table 1
Calculated octahedron angle for iron(III) complexes
Angle 1
Angle 2
Angle 3
Average
Difference with deferiprone
Deferiprone
8a
8b
174.7605
175.2007
176.3598
173.6917
175.1695
176.4442
172.0701
175.2263
176.3909
173.5074
175.1988
176.3983
0
1.6914
2.8909
Table 2
Antimicrobial activity of chelators (MIC)a
Chelators
S. aureus
B. cereus
B. subtilis
g/mL
E. coli
P. aeruginosa
l
g/mL
lM
l
g/mL
lM
l
lM
lg/mL
lM
l
g/mL
lM
1
8a
8b
DTPA
40
40
40
40
56.9
50.8
48.2
40
40
80
80
56.9
50.8
96.4
40
40
80
80
56.9
50.8
96.4
80
40
40
80
113.7
50.8
48.2
24
40
40
80
34.1
50.8
48.2
101.7
203.4
203.4
203.4
203.4
a
MICs for bacteria were determined on BHI agar after 24 h of incubation at 37 °C, according to the standard agar dilution technique and with visual inspection of bacterial
growth.23 The tubes were seeded at a density of approximately 1.0 ꢁ 107 colony-forming units per milliliter (CFU/mL).
limitation resulting from intramolecular links, as the ligands will
position themselves with respect to the central metal ion in order
to achieve maximum bonding overlap for coordination bond for-
mation. In contrast, when joined by a covalent structure, the che-
lating moieties will experience a restrictive inflexibility and will
probably be unable to achieve optimal distribution around the
coordinated metal. Significantly the arm length of 8a provides an
angle (175.2) which is closer to that of deferiprone (173.5) than
that of 8b (176.4).
By using the determined pKa values and log stability constants,
the pFe3+ value, which is defined as the negative logarithm of
concentration of free iron(III) in solution under conditions total
[ligand] = 10ꢀ5 M and total [iron] = 10ꢀ6 M at pH 7.45 was calcu-
lated. The corresponding pFe3+ values of 8a and 8b, under these
conditions, are 30.36 and 30.04, respectively, indicating 8a pos-
sesses a moderately higher affinity for iron(III) than 8b, which is
in agreement with the result of the molecular dynamics simulation
studies of the iron complexes.
The antimicrobial activity of chelators 8a and 8b was evaluated
in direct comparison with that of diethylene-triamine pentaacetic
acid (DTPA). It was found that both chelators demonstrated a
strong inhibitory effect on the growth of the three tested Gram po-
sitive bacteria (Staphyloccocus aureus, Bacillus subtilis and Bacillus
cereus) and the two Gram negative bacteria (Escherichia coli and
Pseudomonas aeruginosa) (Table 2). In the case of S. aureus, the ef-
fect of both 8a and 8b was similar to that of DTPA, with a MIC of
P. aeruginosa, both 8a and 8b exhibited relatively stronger inhibi-
tion than DTPA with a MIC of 40 g/mL. In comparison with 1,
8a exhibited similar inhibitory effect against the three tested Gram
positive bacteria. 8a and 8b showed stronger inhibition than 1
against E. coli, but a relatively weaker inhibitory activity against
P. aeruginosa.
l
Overall, of the chelators investigated, 8a and 8b showed very
similar antimicrobial activity and they were found to possess
stronger antimicrobial activity than DTPA in most cases. This is pri-
marily due to the fact that 8a and 8b possess a higher affinity for
iron(III) than DTPA (pFe = 24.6).24 The calculated partition coeffi-
cients (c log P) of 8a and 8b, obtained by MarvinView 5.4.0.1 soft-
ware,25 were found to be ꢀ2.27 and ꢀ1.56, respectively, this
hydrophilicity together with the relatively high molecular weights
(8a: 641, 8b: 683) would suggest that these complexes will not be
able to cross bilayer membranes by non-facilitated diffusion. Thus
with Gram negative bacteria they may gain access to the periplas-
mic space and there restrict the availability of iron to the bacte-
rium. These chelators may also inhibit the growth of bacteria by
scavenging iron in the environment around the bacteria. 8a and
8b may find application in the treatment of external infections
such as those associated with wounds.
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),
40
lg/mL. Against B. cereus and B. subtilis, 8a showed the strongest
inhibitory activity with a MIC of 40
lg/mL. In the case of E. coli and

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B. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6376–6380
(CH in Ph), 137.6 (C in Ph), 140.3 (C-2 in pyridinone), 145.6 (C-3 in pyridinone),
147.8 (C-6 in pyridinone), 171.2 (C-4 in pyridinone), 172.0 (CONH). ESI-MS:
954 ([M+H]+); HRMS: Calcd for C54H64N7O9 ([M+H]+) 954.4764, found
954.4751.
Scientific Research Foundation for the Returned Overseas Chinese
Scholars, State Education Ministry of China ([2009]1590) and
Qianjiang Scholars Fund, Zhejiang Province (No. 2010R10051).
16. Data for 8a: 1H NMR (DMSO-d6) 2.55 (s, CH3, 9H), 3.55 (br, CH2, 6H), 3.85 (s,
CH3, 9H), 4.62 (d, J = 4.5 Hz, CH2, 6H), 7.33 (s, Pyridinone 5C-H, 3H), 9.29 (br,
NH, 3H); 10.70 (br, 1H, HN+); 13C NMR (DMSO-d6) d 20.6 (CH3), 34.6 (NHCH2-
pyridinone), 39.3 (NCH3), 56.8 (NCH2CO), 112.6 (C-5H in pyridinone), 139.4
(C-2 in pyridinone), 142.6 (C-3 in pyridinone), 148.3(C-6 in pyridinone), 159.3
(C-4 in pyridinone), 211.0 (CONH). ESI-MS: 642 ([M+H]+), 664 ([M+Na]+);
HRMS: Calcd for C30H40N7O9 ([M+H]+) 642.2886, found 642.2883. Data for 8b:
1H NMR (DMSO-d6) 2.58 (s, CH3, 9H), 2.75 (t, J = 7.0 Hz, CH2, 6H), 3.28 (m, CH2,
6H), 3.90 (s, CH3, 9H), 4.62 (d, J = 4.8 Hz, CH2, 6H), 7.35 (s, Pyridinone 5C-H,
3H), 9.01 (t, J = 4.7 Hz, NH, 3H), 11.00 (br, HN+, 1H); 13C NMR (DMSO-d6) d 20.5
(CH3), 28.8 (NCH2CH2), 34.5 (NHCH2-pyridinone), 39.2 (NCH3), 48.2 (NCH2CH2),
112.5 (C-5H in pyridinone), 139.4 (C-2 in pyridinone), 142.6 (C-3 in
pyridinone), 148.3(C-6 in pyridinone), 159.3 (C-4 in pyridinone), 169.3
(CONH). ESI-MS: 684.3 ([M+H]+), 342.7 ([M+2H]2+); HRMS: Calcd for
References and notes
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C
33H46N7O9 ([M+H]+) 684.3356, found 684.3320.
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22. Molecular dynamics simulation method: In order to find a global minimum
structure of hexadentates in the gas phase, simulated annealing approach
using MM+ forcefield (a molecular mechanic method) in HyperChem 8.0
(http://www.hyper.com/) was undertaken. A starting structure was simulated
at 1000 K for a period of time (randomly between 0.1 ps and 1 ps) and then
quenched to an energy minimised structure. This procedure was repeated for
48,000 times and the structure with the lowest potential energy was recorded.
There are four types of starting structure, because the tertiary nitrogen can
adopt two conformations (amine lone pair electrons oriented towards Fe3+ and
away from Fe3+) and the chelating groups can adopt two conformations (left-
hand propeller and right-hand propeller isomers). All the hexadentates studied
here were constructed in these four starting structures and each processed by
the same procedure.
15. Data for 7a: 1H NMR (DMSO-d6) 2.22 (s, CH3, 9H), 3.30 (s, CH2, 6H), 3.34 (s, CH3,
9H), 4.35 (d, J = 5.0 Hz, CH2, 6H), 5.07 (s, CH2, 6H), 6.18 (s, Pyridinone 5C-H,
3H), 7.29–7.35 (m, Ph, 9H), 7.42 (m, Ph, 6H), 8.39 (t, J = 5.0 Hz, NH, 3H); 13C
NMR (DMSO-d6) d 20.0 (CH3), 34.1 (NHCH2-pyridinone), 35.6 (NCH3), 57.4
(NCH2CO), 72.1 (CH2Ph), 117.4 (C-5H in pyridinone), 127.7 (CH in Ph), 128.1
(CH in Ph), 128.3 (CH in Ph), 137.6 (C in Ph), 140.0 (C-2 in pyridinone), 145.6 (C-
3 in pyridinone), 147.7 (C-6 in pyridinone), 170.5 (C-4 in pyridinone), 171.9
(CONH). ESI-MS: 912 ([M+H]+); HRMS: Calcd for C51H58N7O9 ([M+H]+)
912.4295, found 912.4270. Data for 7b: 1H NMR (DMSO-d6) 2.18 (m, CH2,
6H), 2.25 (s, CH3, 9H), 2.60 (m, CH2, 6H), 3.37 (s, CH3, 9H), 4.33 (d, J = 4.9 Hz,
CH2, 6H), 5.07 (s, CH2, 6H), 6.17 (s, Pyridinone 5C-H, 3H), 7.28–7.36 (m, Ph, 9H),
7.42 (m, Ph, 6H), 8.13 (t, J = 4.9 Hz, NH, 3H); 13C NMR (DMSO-d6) d 20.0 (CH3),
32.8 (NCH2CH2), 34.3 (NHCH2-pyridinone), 35.6 (NCH3), 48.9 (NCH2CH2), 72.2
(CH2Ph), 117.4 (C-5H in pyridinone), 127.8 (CH in Ph), 128.2 (CH in Ph), 128.3
23. Barry, A. L. The Antimicrobic Susceptibility Test: Principles and Practices In
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25. http://www.chemaxon.com