The influence of linkages between 1-hydroxy-2(1H)-pyridinone coordinating groups and a tris(2-aminoethyl)amine core in a novel series of synthetic hexadentate iron(III) chelators on antimicrobial activity

Article abstract of DOI:10.1016/j.bioorg.2019.103465

Resistance of pathogens to antimicrobials is a major current healthcare concern. In a series of linked studies, we have investigated synthetic iron chelators based on hydroxy-pyridinone ligands as novel bacteriostatic agents. Herein we describe our synthesis of several useful building blocks based on the 1-hydroxy-2(1H)-pyridinone moiety, including a novel formyl derivative, which were combined with a tris(2-aminoethyl)amine core to obtain a series of new high-affinity hexadentate Fe(III) chelators. The design principle examined by this series is the size and flexibility of the linker between the core and the metal ligands. Measurement of the pKa and stability constants (Fe³⁺ and Cu²⁺) of representative coordinating groups was performed to help rationalise the biological activity of the chelators. The novel chelators were tested on a panel of representative microorganisms with some effectively inhibiting microbial growth. We demonstrate that the nature and position of the linker between the hydroxypyridinone and the tris(2-aminoethyl)amine core has considerable impact upon microbial growth inhibition and that both amide or amine linkages can give efficacious chelators.

Full text of DOI:10.1016/j.bioorg.2019.103465

Journal Pre-proofs
The influence of linkages between 1-Hydroxy-2(1H)-pyridinone Coordinating
Groups and a Tris(2-aminoethyl)amine core in a novel series of synthetic
Hexadentate Iron(III) Chelators on antimicrobial activity
David G. Workman, Michael Hunter, Shuning Wang, Jérémy Brandel,
Véronique Hubscher, Lynn G. Dover, David Tétard
PII:
S0045-2068(19)30983-6
DOI:
https://doi.org/10.1016/j.bioorg.2019.103465
YBIOO 103465
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
19 June 2019
30 September 2019
21 November 2019
Please cite this article as: D.G. Workman, M. Hunter, S. Wang, J. Brandel, V. Hubscher, L.G. Dover, D. Tétard,
The influence of linkages between 1-Hydroxy-2(1H)-pyridinone Coordinating Groups and a Tris(2-
aminoethyl)amine core in a novel series of synthetic Hexadentate Iron(III) Chelators on antimicrobial activity,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.103465
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The influence of linkages between 1-Hydroxy-2(1H)-pyridinone Coordinating Groups and a Tris(2-
aminoethyl)amine core in a novel series of synthetic Hexadentate Iron(III) Chelators on
antimicrobial activity.
a
a
b,c
b,c
b,c
David G. Workman , Michael Hunter , Shuning Wang , Jérémy Brandel , Véronique Hubscher , Lynn
a
a
G. Dover , David Tétard *
a
Faculty of Health and Life Sciences, Northumbria University, Newcastle upon Tyne, NE1 8ST, United
Kingdom.
b
Université de Strasbourg, IPHC, 25 rue Becquerel 67087 Strasbourg, France
c
CNRS, UMR7178, 67087 Strasbourg, France
Corresponding author: david.tetard@northumbria.ac.uk. Phone: Int + 44 (0)191 227 4934
Summary
Resistance of pathogens to antimicrobials is a major current healthcare concern. In a series of linked
studies, we have investigated synthetic iron chelators based on hydroxy-pyridinone ligands as novel
bacteriostatic agents. Herein we describe our synthesis of several useful building blocks based on the 1-
hydroxy-2(1H)-pyridinone moiety, including a novel formyl derivative, which were combined with a tris(2-
aminoethyl)amine core to obtain a series of new high-affinity hexadentate Fe(III) chelators. The design
principle examined by this series is the size and flexibility of the linker between the core and the metal
ligands. Measurement of the pKa and stability constants (Fe3+ and Cu ) of representative coordinating
groups was performed to help rationalise the biological activity of the chelators. The novel chelators were
tested on a panel of representative microorganisms with some effectively inhibiting microbial growth. We
demonstrate that the nature and position of the linker between the hydroxypyridinone and the tris(2-
aminoethyl)amine core has considerable impact upon microbial growth inhibition and that both amide or
amine linkages can give efficacious chelators.
2+
Keywords
1

Siderophore, Chelator, Biostatic agent, Bacterial Growth, 1-Hydroxy-2(1H)-pyridinone
1- Introduction
A major public health concern is the loss of activity of once efficacious antibiotics due to emerging
bacterial resistance and its transfer among bacterial populations. The need to develop new antibiotics and
new strategies to combat the spread of multi-drug resistant infections is pressing. It has long been known
that metal chelators impose biostasis upon microorganisms. Their mode of action is thought to be the
sequestration of the metal and subsequent starving of the microorganisms.1-6
For its biological importance, iron is an ideal primary target for metal withdrawal. In the case of infections,
iron is plentiful in the body of the patient but is not readily available to the microorganism. The pathogen
must compete with its host to mobilise iron.7-9 Pathogenic microorganisms have evolved efficient iron
acquisition mechanisms, the most remarkable one being based on small Fe3+ chelating molecules called
siderophores, of which there are hundreds of examples.10-11 The host employs various Fe3+ withholding
strategies to limit bacterial growth including the production of the protein siderocalin that interferes with
the siderophore-based iron acquisition.12-14
The majority of siderophores are hexadentate chelators; their Fe3+ affinities/binding strengths (as
measured by pFe3+15 can be very high, in excess of 25. They are thus able to acquire otherwise
unavailable iron from sequestered host sources.16-19 To complement the action of siderocalin in inhibiting
siderophore -mediated iron uptake by pathogens, it is possible to envisage the therapeutic use of a
synthetic chelator acting in the growth medium.
*
Alongside the difficulties associated with the design and synthesis of powerful therapeutic chelators is the
risk of denaturation of essential metalloenzymes in the body of patients.20-22 This and other factors
complicate the development of anti-infective chelators. Further research and understanding of the effect
*
pFe3+ is defined as -log[Fe3+]free, usually calculated at pH 7.4, with [Chelator]total = 10 M and [Fe3+]total
M and cited herein in these conditions.
=
1
2

of the structure of biostatic chelators on the thermodynamics and kinetics of binding of all relevant
biological metals and their reactivity with human metalloenzymes are required.
The focus of our research is on the design of chelators for powerful Fe3+ chelation and competition with
the high affinity iron uptake systems of pathogens, including siderophores, to complement the activities of
antimicrobial compounds. To compete with bacterial siderophores (that are commonly based on
coordinating groups such as -hydroxycarboxylic acids, hydroxamic acids and catechols)11, 18, it is crucial
to select the right coordinating group. Focusing purely on thermodynamic aspects of metallation in
competition between siderophores and another chelator, the respective pFe3+ of the two molecules is the
key parameter to consider.23 The pFe3+ value encompasses all the factors influencing the chelation of
3+
Fe , including the structural parameters, pKa and stability constant(s). It would appear that three isomers
of the bidentate hydroxypyridinone (HOPO) family (Figure 1) often endow hexadentate chelators with a
good combination of pKa and log(Fe ) and therefore generate sufficiently high pFe values.19
3
+
3+
O
O
O
OH
OH
H
N
OH
N
N
H
1
-Hydroxy-2(1H)-pyridinone
3-Hydroxy-4(1H)-pyridinone
(3,4-HOPO)
3-Hydroxy-2(1H)-pyridinone
(3,2-HOPO)
(1,2-HOPO)
Figure 1. Three relevant isomers of the HOPO family.
Additional attractive properties can be expected through the use of HOPO coordinating groups. It is well
known that many bacteria can exploit siderophores elaborated by other microbial species, a process
9
called “siderophore piracy”. In the proposed application, any synthetic chelator must not be recognised
and taken up by pathogens to promote their growth.6, 24 As very few HOPO are known in natural
compounds and only one of the 1,2-HOPO class has been described as a siderophore, (the bidentate 1-
hydroxy-5-methoxy-6-methyl-2(1H)-pyridinone also called cepabactin)25 it is unlikely that metallated
3+
HOPO-based chelators will be recognised by bacterial receptors and exploited as a source of Fe , but
not impossible.26 Furthermore, a therapeutic chelator must not interact with siderocalin either and thus
inhibit its protective capacity. The binding between some 1,2-HOPO-based chelators and siderocalin has
3

been studied and was found to be very weak, suggesting that this class of coordinating groups would not
overwhelm this defence strategy, but rather may complement it.27
The most powerful Fe3+ chelator of the HOPO family is 3,4-HOPO.28 Several hexadentate chelators
based on that coordinating group, are also known to have antimicrobial action.29-39 Interestingly, the
opposite effect, i.e. promotion of the growth of Escherichia coli by an Fe3+ complex of a synthetic
hexadentate 3,4-HOPO chelator has been observed26 highlighting the need to fully understand the
relationship in these chelators between structure and biological function.
We have recently described the first examples of biostatic hexadentate chelators based on 1,2-HOPO.40
We considered that this class of HOPO may have advantages over the 3,4-HOPO isomers despite being
predicted to be slightly weaker Fe3+ chelators.28 For example, a lower pKa of the hydroxyl group in 1,2-
HOPO may help limit host cell penetration and therefore may have advantages with respect to patient
safety. To the best of our knowledge, until our first paper, only a very small number were known to
contain at least one 1,2-HOPO moiety and these are illustrated in Figure 2.27, 41-44
4

O
OH
HN
O
N
OH
O
N
O
H
N
O
N
HN
N
OH
O
H
N
N
OH
NH
O
OH
O
1
O
NH
HO
N
2
HO
HO
O
O
OH
N
O
HN
H
N
N
N
O
O
OH
O
O
N
NH
N
3
OH
HO
O
O
OH
N
O
HN
N
O
HN
H
N
N
O
N
N
O
HO
NH
N
O
N
NH
O
OH
O
H
O
HO
O
4
N
N
5
HO
O
Figure 2. Hexadenate chelators based on 1,2-HOPO reported in the literature before Workman et al. . 1 see ref 42; 2
40
see ref 27; 3 see ref 43; 4 see ref 44; 5 see ref 41
.
Previously, our primary focus was to study the effect of the molecular backbone on biostatic activity rather
than that of the linker between the 1,2-HOPO moiety and the backbone. Our promising results using a
methylene linker between a 1,2-HOPO group and the 1,4,7-triazacyclonone backbone40 prompted us to
5

investigate further the effect of linker structure in order to identify the most promising biostatic agents for
further physicochemical and mechanistic studies. Of the previously known 1,2-HOPO-based hexadentate
chelators on Figure 2, the focus is almost systematically on amide linkage on position 6 of the 1,2-HOPO
ring. Studying other types of linkers may facilitate optimisation of the biostatic agent. A new hexadentate
1
,2-HOPO-based chelator using a methylene linker was recently published by others.45
Here we report the synthesis of a range of hexadentate chelators bearing three 1,2-HOPO as
coordinating groups anchored on a tris(2-aminoethyl)amine (TREN) core (Figure 3). This core has been
used frequently in the synthesis of various hexadentate iron chelators based on HOPO and therefore
selected here because it provides a common feature useful when comparing extant compounds.29, 43, 45-47
Primarily, our novel chelators have been designed to explore novel systematic structural variations of the
linkages between TREN and the hydroxypyridinone moiety, in particular secondary and tertiary amides,
secondary and tertiary amines. Additionally, we show the effect of these chelators upon microbial growth.
The contributory effect the linkages have on the chelation properties of the molecules i.e. pKa of the
hydroxyl group, stability constants, will be reported separately.
6

O
O
OH
OH
N
N
N
N
NH
N
H
N
N
N
HN
O
N
O
N
HO
O
OH
HO
O
OH
N
N
7
6
O
OH
OH
O
N
N
O
O
N
N
NH
N
H
N
O
N
N
N
N
HN
O
N
N
O
HO
HO
O
OH
O
O
O
9
8
O
OH
OH
N
O
O
NH
N
H
N
N
HN
O
N
HO
O
O
O
OH
10
Figure 3. Novel chelators synthesised.
2- Results and Discussion
2.1 Synthesis
7

The access to our targeted chelators relied primarily on two key novel building blocks that were already
available in our laboratory.48 Firstly, starting from 2-hydroxy-pyridine-6-carboxylic acid 11 building block
12, i.e. 1-allyloxy-6-hydroxymethyl-2(1H)-pyridinone was easily obtained in four steps. Starting from 2-
chloro-pyridine-3-carboxylic acid (13) the second key building block 14, i.e. 1-hydroxy-2(1H)-pyridinone-3-
carboxylic acid was also easily obtained in two steps from (Scheme 1).
The choice of an allyl protective group of the 1-hydroxyl group of building block 11 and subsequent
derivatives requires explanation. In order to expand the existing 1,2-HOPO chemistry to incorporate
methylene-linked binding units into target multi-dentate chelate molecules (see below), a novel HOPO
protection strategy instead of the traditionally used benzyloxy protection for 1,2-HOPO was required.
Specifically, it was predicted that benzyl protection would not be suitable as the most common methods
used for its removal would probably also cleave the linkage between our HOPO group and the molecular
backbone. Of the protective methods identified, protection of the N-hydroxyl group as the allyloxy group
was chosen. It was considered that deprotection could be achieved using a range of relatively mild
conditions, for example via double bond isomerisation and subsequent hydrolysis without compromising
other bonds in the chelator.49
(a) - 4 steps
(b)
Cl
OH
O
N
O
O
N
O
HO
N
CO H
2
11
12
(c)
15
CO H
2
CO H
2
(d) - 2 steps
O
N
O
CHO
N
O
N
Cl
OH
16
13
14
Scheme 1. (a) 4 steps: (i) CH
3
CO
3
H, acetic acid, heat (77%), (ii) SOCl
2
, methanol, heat (96%), (iii) allyl bromide,
, dichloromethane, heat (92%) (c)
3 3 3
, dichloromethane, 0 C (33%) (d) 2 steps: (i) CH CO H, acetic acid, heat (42%) (ii) KOH,
K
2
CO , acetonitrile, heat (94%), (iv) NaBH
3
4
, methanol, THF, heat (65%) (b) SOCl
2
o
oxalyl chloride, DMSO, NEt
water, heat (92%).
8

The hydroxymethyl building block 12 could be converted to the novel chloromethyl derivative 15 and
aldehyde 16 for incorporation onto the ligand cores. Novel isomer 14 of the original 1-hydroxy-2(1H)-
pyridinone-6-carboxylic acid was required to assess any effect the regiochemistry has on the biological
activity, with respect to the linker positioning that could have an impact on the pKa and metal stability
constants (see below). With these novel building blocks in hand, it was then possible to build a more
systematic series of hexadentate chelators based on 1,2-HOPOs.
Access to a more-flexible analogue of 3, namely compound 6 (Scheme 2), where the carbonyl linking unit
was replaced by a methylene group, was prepared following reductive amination of TREN (17) by
aldehyde 16 in the presence of sodium borohydride, affording the triallyl-protected chelator 18 in poor
yield (15%). Despite this yield, that method was preferable to direct alkylation of TREN using 15, which
resulted in an even lower yield (9%) attributed to excess alkylation of the amine pendant arms.
O
OR
N
NH2
N
NH
N
(a)
3
O
N
O
CHO
+
H
H N
2
NH2
N
HN
N
O
16
17
RO
O
OR
N
1
8, R = allyl
(b)
6
, R = H
(isolated as mono HCl salt)
Scheme 2. Reagents and conditions: (a) (i) TREN, ethanol, heat, (ii) NaBH
4
(15%) (b) BCl , dichloromethane
3
(75%).
9

Although removal of the allyl group using a HCl/AcOH mixture (a treatment that would be used to remove
a benzyl protection) was possible, it also resulted in unacceptably frequent cleavage of the HOPO moiety
from the TREN backbone. This problem was initially predicted and justified the need in our strategy for
early identification of a different non-benzyl protective group. Attempts to deprotect 18 using Pd/C +
TFA/1,4-dioxane 50 did not give chelator 6. This was attributed to poisoning of the catalyst due to the
presence of secondary amine groups. However, de-allylation of 18 using boron trichloride was successful
and produced the novel secondary amine 6 in good yield (75%). As expected, an allyl protective group
therefore proved very useful as at it was removed without cleavage of the methylene linker.
To further illustrate the versatility of the new building blocks, a tertiary amine linked chelator was
designed. TREN was converted into tricarbamate 19 that was then reduced to a new backbone tris(N-
3
methyl-2-aminoethyl)amine, or (Me) TREN 20, by a previously reported procedure as shown in Scheme
3
.51
EtO
O
NH2
N
NH
NH
N
(a)
(b)
N
H
H
H N
2
NH2
O
N
HN
OEt
N
HN
OEt
O
17
19
20
2 4
Scheme 3. Reagents and conditions: (a) ClCO Et, KOH, toluene, 0°C (crude 92%) (b) (i) LiAlH , THF, heat (ii) KOH
(19%).
Synthesis of 7 continued by the alkylation of 20 by chloromethyl 1,2-HOPO 15, albeit in low yield (35%)
Scheme 4). Allyl removal from compound 21 using Pd/C catalyst in a 1,4-dioxane/TFA mixture afforded
(
the novel chelator 7 in good yield (66%), without noticeable catalyst poisoning, again demonstrating the
usefulness of using the allyl protective group in our synthetic strategy.
10

O
OR
N
N
N
NH
N
(a)
Cl
O
N
O
3
+
H
N
N
N
HN
N
O
RO
O
OR
15
N
20
2
1, R = allyl
(b)
7
, R = H
(isolated as the tetra-trifluoroacetic acid salt)
Scheme 4. Reagents and conditions: (a) K
2
3
CO , acetonitrile, heat (35%) (b) Pd/C, TFA (0.17 M), 1,4-dioxane/water,
heat (66%).
To complete the series of secondary and tertiary amines and amides necessary for biological testing, the
synthesis of 3 was conducted as previously described52 and the novel chelator 8 was synthesised as
described in Scheme 5. Activation of known compound 22 as the acid chloride and subsequent coupling
with 20 resulted in a poor yield. Instead, activation of 22 as the pentafluorophenol ester 23 was
performed53 and coupling with 20 gave 24 in a moderate yield (58%). Deprotection of 24 in a strong acid
mixture afforded the desired novel chelator 8 in excellent yield (90%).
11

O
OR
N
O
N
N
(a)
(b)
O
N
CO H
O
N
CO C F
2
2
6
5
O
N
N
N
OBn
OBn
N
O
RO
O
O
OR
22
23
2
4, R = Benzyl
(c)
8
, R = H
(isolated as the hydrochloride
salt)
i
Scheme 5. Reagents and conditions: (a) CF
3
CO
2
C
6
F
5
, pyridine, DMF (b) 20, (Pr)
2
NEt, DMF (58%) (c) HCl:AcOH
(90%).
52
Following a slight modification of a method previously described by Raymond et al. , novel chelator 9,
the retro analogue of chelator 3 was synthesised (Scheme 6). Compound 14 was first benzyl-protected
into 25 that was then converted to the mercaptothiazoline-activated derivative 26. EDC (1-ethyl-3-(-3-
dimethylaminopropyl)carbodiimide hydrochloride) was used instead of using DCC as the dehydrating
agent in that latter step as we found removal of the reaction by-products was more easily accomplished.
Coupling of 26 with TREN gave 27 that was deprotected in strongly acidic conditions (HCl:AcOH) to give
chelator 9. To the best of our knowledge, 9 is the first analogue of previously known hexadentate chelator
3
where the attachment point is in position 3 on the 1,2-HOPO ring.
12

OR
N
O
O
O
O
S
NH
N
CO H
(a)
2
(b)
(c)
N
14
S
N
O
N
H
N
N
HN
O
N
OBn
OBn
RO
26
25
O
O
O
OR
2
7, R = Benzyl
, R = H
isolated as the hydrochloride salt)
(d)
9
(
2 3
Scheme 6. Reagents and conditions: (a) benzyl bromide, K CO , methanol, heat (85%) (b) 2-Mercaptothiazoline,
EDC, 4-(N,N-dimethylamino)pyridine (cat.), dichloromethane (44%) (c) TREN, dichloromethane (77%) (d)
HCl:AcOH, 50°C (82%).
Because 1,2-HOPO moieties are isoelectronic to catechol (a frequently found coordinating group in
bacterial siderophores), we considered that some of this series of chelators might be recognised by
bacterial ferri-siderophore receptors akin to “siderophore piracy” (as previously shown for synthetic tris-
catechol synthetic chelators). An undesirable effect of such recognition might be the promotion of
bacterial growth.54 We therefore sought analogues in which the 1,2-HOPO group would not resemble
natural catechols. Two more analogues of compound 3 were synthesised containing fused benzene rings
on the HOPO moiety. Besides its possible effect on the pKa and stability constant for Fe3+ (hence on
3+
pFe ), the extra aromatic ring might inhibit recognition of the ferric chelator by bacterial receptors.
Compound 1 was synthesised as described previously.55 A retro analogue of chelator 1, namely
compound 10 was also prepared from 1,2-HOPO derivative 28 (Scheme 7). Although compound 28 has
long been known,56 it was never been used in the synthesis of hexadentate metal chelators. Using the
same method as for the synthesis of compound 1, novel chelator 10 could be obtained in four steps from
28.
13

CO H
CO H
2
2
(a)
(b)
N
O
N
O
OH
OBn
OR
N
28
29
O
O
NH
N
COCl
O
(c)
H
N
N
N
HN
O
N
RO
OBn
O
O
O
30
OR
3
1, R = Benzyl
0, R = H
isolated as the hydrochloride salt)
(d)
1
(
Scheme 7. Reagents and conditions: (a) Benzyl bromide, K
2
3
CO , methanol, heat (83%) (b) Oxalyl chloride, DMF
(
cat.), toluene, 40°C (51%) (c) TREN, NEt , toluene (65%) (d) HCl:AcOH, 50°C (57%).
3
To test our hypothesis that chelator 3 and 9 limit the growth of bacteria via chelation in the growth
medium rather than through other mechanisms linked to the TREN core after cell uptake, close
analogues of these compounds where the coordinating groups were partially removed were considered
desirable. Thus control compounds 32 and 33 were prepared as shown in Scheme 8.
14

-O
N+
O
NH
(a)
N
N+ CO H
H
2
N
HN
O
O-
N+
O-
O
O-
N+
32
HO
N
O
NH
(b)
N
N
CO H
H
2
N
N
HN
O
OH
OH
O
OH
N
33
3 3
Scheme 8. Reagents and conditions: (a) HOBt, EDC, NEt , TREN, dichloromethane (32%) (b) TREN, PPh ,
(C
5
H
5
NS) , acetonitrile, 60°C (34%).
2
2.2 Physicochemical Properties
In order to assess the relative intrinsic metal binding affinity of the 1,2-HOPO coordinating group in
compound 3 and 9 (positional isomers) without the thermodynamic influence of the backbone and without
intramolecular hydrogen bonding and to assess their efficacy compared to 3,4-HOPO, their N,N-dimethyl
amide analogues 34 and previously known 3557 were synthesised (Scheme 9). Their pKa and the stability
constants of their Fe3+ and Cu2+ complexes were determined by potentiometric and spectrophotometric
titrations.
15

O
O
O
2
steps
OH
N
N
O
N
N
O
N
OH
O
OBn
OH
35
25
34
o
3 2 2
Scheme 9. Synthesis of 34. (i) oxalyl chloride, DMF (cat.), 60 C then (CH ) NH (71%); (ii) H , Pd/C, HCl, Ethanol,
Water (94%).
The spectrophotometric titration of ligands 35 versus pH (Figure 4) showed one band at 286 nm under
very acidic pH (p[H]=-0.75, the brackets indicate that the values were not measured but calculated from
+
p[H] = -log[H ]). This band underwent a first bathochromic shift to 303 nm, then a second one to 324 nm.
These variations suggest the presence of three species over the whole studied pH range, (35)H
and 35.
2
+, (35)H
2
1
0
.5
2
p[H] -0.75
1
.8
.6
.4
.2
0
p[H] 1.75
0
0
0
0
pH 1.93
pH 12.45
.5
1
.5
0
2
20
270
320
(nm)
370
240
290
340
390


(nm)
a)
Figure 4. Spectrophotometric titration versus pH of ligand 35 a) between p[H] -0.75 and 1.75 (batch), [35] = 3.08×10-4
M, and b) between pH 1.93 and 12.45, [35] = 1.18×10-4 M; I = 0.1 M (NaClO
b)
4
2
); T = 25.0(1)⁰C; solvent: H O.
The fitting of the data allowed us to determine the acidity constants (pKa=- log K) of the two equilibria
Table 1) and to propose the protonation scheme (Scheme 10).
(
16

K1
K2
N
N
N
O
N
O-
O
N
HO
N+
O
OH
O
OH O
Scheme 10. Protonated species of ligand 35.
Ligand 34 showed also one equilibrium in the 2-12 pH range (Figure S1b). Under very acidic pH, the
hypochromic shift of the last two spectra suggested that the second protonation constant would be at
much lower p[H] values (Figure S1a). The protonation constants allowed us to calculate the electronic
spectra and the distribution curves of the protonated species of ligands 34 (Figure S2) and 35 (Figure 5),
-
which showed that the ligands are in their totally deprotonated form L (L= 34, 35) at physiological pH (pH
7.4).
8
6
4
2
000
(
35)H2
(
35)H
100
(
35)
(35)H
(35)
000
000
000
0
38
8
0
60
4
2
0
0
0
(
35)H2
2
40
290
340
-
0.75 1.25 3.25 5.25 7.25 9.25 11.25

(nm)
pH
a)
b)
Figure 5. a) Electronic spectra and b) distribution curves ([35] = 2.5×10-4 M) of the protonated species of ligand 35. I
0.1 M (NaClO ); T = 25.0(1)⁰C; solvent: H O.
=
4
2
The hydroxyl group of chelator 35 is slightly more acidic than that of chelator 34. This can probably be
explained by the electronic effect of the substituent of the 6- and 3- position respectively and the
existence of a bifurcated hydrogen bond in chelator 35.
The complexation of Fe(III) by ligands 34 (Figure 5) and 35 (Figure S3) was then studied
spectrophotometrically over the same pH range than the protonation studies. It showed for both ligands
the appearance of a ligand-to-metal charge transfer band (LMCT) from very low pH that shifted
hypsochromically when the pH increased up to around pH 6. Then, the LMCT band gradually decreased
17

and the baseline started increasing (see Figure 5b at 580 nm), sign that Fe(III) is released from the ligand
and precipitating as Fe(OH)
3
.
0
0
0
0
.4
.3
.2
.1
0
pH 5.78
0
0
0
.3
.2
.1
0
p
[H] 2
pH 2.03
pH 12.25
p
[H] -0.75
3
70
420
470
 (nm)
520
570
3
70
420
470
520
570

(nm)
a)
b)
Figure 6. Spectrophotometric titration versus pH of ligand 34 + Fe(III) ([34]/Fe(III) = 3.2) a) between p[H] -0.75 and
2
.0 (batch), [34] = 3.01×10-4 M, and b) between pH 2.03 and 12.25, [34] = 2.34×10-4 M; I = 0.1 M (NaClO
5.0(1)⁰C; solvent: H O.
4
); T =
2
2
2 3
Data analysis allowed us to highlight the consecutive formation of FeL, FeL and FeL species for both
ligands and their overall stability constants were calculated (Table 1). From these values, we calculated
the electronic spectra and distribution curves of the complexes of ligands 34 (Figure 7b) and 35 (Figure
S4) versus pH.
1
00
Fe(OH)3
Fe(34)2
4
3
2
1
000
000
000
000
0
Fe(34)3
Fe(34)2
Fe(34)
Fe(34)3
5
0
0
Fe(34)
3
60
410
460
(nm)
510
560
0
2
4
6
8
10
12

pH
a)
b)
Figure 7. a) electronic spectra and b) distribution curves of the Fe(III) complexes of ligand 34 ([34] = 2.5×10-4 M,
34]total/[Fe]total = 3.2); I = 0.1 M (NaClO ); T = 25.0(1)⁰C; solvent: H O.
[
4
2
18

The distribution curves of both ligands showed that, at physiological pH, Fe(III) would already be partially
released from the bidentate ligands (25% Fe(34) , 75% Fe(OH) ), what confirms the need to design
hexadentate ligands by anchoring three bidentate units together.
3
3
Titrations of both ligands with Fe(III) were carried out at pH 2 (Figure S5) as well as competition
experiments with EDTA in Hepes buffer at pH 7.4 (Figure S6). The conditional stability constants at fixed
pH obtained from these experiments were converted to overall stability constants and were in good
agreement with the one obtained from the titrations versus pH (Tables S1 and S2), confirming our
complexation models. The complexation ability of both ligands with Cu(II) was studied by potentiometry
2
and showed the formation of CuL and CuL for both ligands.
Table 1. Protonation constants (pKa) of ligands 34 and 35, overall stability constants (logmlh) of their
Fe(III) and Cu(II) complexes.
Fe3+
Cu2+
Ligand
pKa
log110
9.59(1)
9.99(2)
log120
18.06(1)
18.66(2)
log130
24.59(3)
25.88(3)
log110
6.2
6.3
log120
11.0
11.6
log121
log12-1
3
3
4
5
5.40(3)
5.20(3)
/
/
/
/
5
7
57
(
5.17 )
0.4
5.78
0.8
9.82
.64
(25.6 )
~
1
,2-HOPOa
10.6
20.1
27.2
7.29(2)
13.06(3)
19.09(1)
/
/
-
DFPb
15.01(1)
27.3(1)
37.43(1)
10.42(2)
21.98(3)
8.49(3)
3
o
T = 25.0 C; solvent : water, I = 0.1 M (NaClO
a. ref
b. ref
4
);
5
8
9
5
In order to compare the chelation power of the different ligands and their selectivity, the pM values were
calculated from the overall stability constants and plotted against pH (Figure 8).
19

M-1,2HOPO
M-DFP
Fe3+
3
2
2
1
1
1
6
1
6
1
6
pH 7.4
M-[34]
M-[35]
M-[OH]
Cu2+
2
3
4
5
6
7
8
9
10
11
12
pH
Figure 8. Chelation power (represented by the pM value) of the ligands versus pH. pM = -log[M]free, [L]total = 10-5 M,
M]total = 10-6 M.
[
For a ligand to dominate metal chelation in aqueous media it must produce a pM curve (M= Fe(III), Cu(II))
above that of the hydroxide anion. The larger the difference, the stronger the chelator.
between pH 2 and 5, ligand 35 and 1,2-HOPO are slightly better chelators than ligands 34 and DFP.
From pH 6.3, DFP, based on the 3,4-HOPO chelating unit becomes a better chelator due to its higher
pKa values. The calculated pCu2+ values showed that, as expected for 1,2-hydroxypyridinones bearing
hard O,O-donors, the affinity for Fe3+ is much higher than for Cu . These results suggested that these
coordinating groups are suited for selective iron(III) chelation and that the synthesised hexadentate (tris-
bidentate) chelators based on ligands 34 and 35 would achieve strong Fe(III) chelation and may primarily
disrupt bacterial iron homeostasis.
2+
2.3 Biological Activity
20

Broth microdilution tests were undertaken based upon a standard procedure.60 in order to obtain
minimum inhibitory concentrations (MICs, Table 2) for the synthetic ligands upon a range of Gram-
negative and Gram-positive bacteria associated with HAIs, as well as the dimorphic fungus Candida
albicans,. The commercially available DTPA (N,N’,N’,N”,N”-diethylenetriaminepentaacetic acid) and
powerful synthetic Fe3+ chelator EMECAM (1,3,5-tris(2,3-dihydroxybenzamidomethyl)-2,4,6-
triethylbenzene, pFe3+ = 32.6)61-62 were also assayed for comparison. Finally, TACN-MeHP (1,4,7-tris(3-
hydroxy-6-methyl-2-pyridylmethyl)-1,4,7-triazacyclononane, Figure 9), the most powerful Fe3+ chelator
known (pFe3+ = 39.4) was used as an additional benchmark.63
OH
N
N
N
HO
N
N
N
HO
TACN-MeHP
Figure 9. 1,4,7-tris(3-hydroxy-6-methyl-2-pyridylmethyl)-1,4,7-triazacyclononane (TACN-MeHP).
21

Table 2. Minimum Inhibitory Concentrations (MIC) expressed in M (in mg/L between brackets) for the ligands tested upon a panel of microorganisms. Results
were determined in triplicate. The molar mass (g/mol) of each chelator is given between brackets in the first column.
Minimum Inhibitory Concentration / M (mg/L)
Chelator
(
molar mass/g mol-
Gram-negative bacteria
Gram-positive bacteria
Fungus
1)
E. coli
DSM-
8039
2542
(1000)
1250
(930.2)
625
(348.5)
625
(345.0)
1250
(1267)
5000
(3180)
2500
(1485)
1250
(930.2)
>5000
(>2547)
>5000
K.
B. subtilis
DSM-
23778
159
(62.5)
625
(465.1)
313
(174.5)
313
(172.8)
625
(633.6)
625
(397.5)
313
(185.9)
S. aureus
DSM-
1104
79
(31.1)
313
(232.9)
5
(2.8)
313
(172.8)
625
(633.6)
625
(397.5)
5
(3.0)
P. aeruginosa
DSM-19880
A. baumannii
DSM-30007
C. albicans
DSM-1386
pneumoniae
DSM-30104
2542
1
DTPA
393.35)
>2542
(> 1000)
313
(232.9)
313
(174.5)
313
(172.8)
625
(633.6)
5000
(3180)
2500
(1485)
625
(465.1)
>5000
(>2547)
>5000
(> 2877)
1928
2542
(1000)
78
(58.0)
313
(174.5)
313
(172.8)
313
(317.3)
1250
(795.1)
313
(185.9)
313
(232.9)
>5000
(>2547)
>5000
(> 2877)
1928
318
(125.1)
313
(232.9)
313
(174.5)
156
(86.1)
78
(79.1)
156
(99.2)
313
(185.9)
625
(465.1)
>5000
(>2547)
>5000
(> 2877)
>5000
(>3289)
39
(
(1000)
1250
(930.2)
313
(174.5)
625
(345.0)
2500
(2534)
>5000
(> 3180)
5000
1
(
b)
(
(
(
744.16 )
3
(b)
593.98 )
6
(b)
552.03 )
7
(c)
(
1013.76 )
8
(b)
(
636.06 )
9
(b)
(
593.98 )
(2969.9)
1250
10
625
156
(b)
(
744.16 )
(930.2)
>5000
(>2547)
>5000
(> 2877)
1928
(1268)
313
(154.2)
(465.1)
>5000
(>2547)
>5000
(> 2877)
1928
(1268)
78
(38.4)
(116.1)
>5000
(>2547)
>5000
(> 2877)
482
(317.0)
78
(38.4)
32
(
509.52)
3
575.47)
(
a)
3
(
(> 2877)
3855
(2536)
78
(38.4)
EMECAM
657.72)
TACN-MeHP
492.63)
(
(1268)
625
(307.9)
(1268)
156
(76.9)
(
(19.2)
(a) Due to solubility issues, the chelator was prepared by addition of three molar equivalents of aqueous sodium hydroxide before solvent removal and it was
evaluated as the tri-sodium salt. (b) as HCl salt. (c) as tetra-trifluoroacetic acid salt.
22

Although the concept used in the design was for a series of synthetic chelators that mediated biostasis by
a competitive extracellular iron limitation, it is possible that this series of compounds may enter the
microbial cell and exert their effects by other mechanisms. The control compounds 32 and 33 were
designed to highlight any biological activity of the TREN backbone and/or of the aromatic rings in
compound 3 and 9 unconnected to their metal chelation activity if the compounds were nevertheless
capable of penetrating the microorganisms. When tested, these control compounds caused no inhibition
of bacterial growth at the concentrations used across the panel of microorganisms. This lack of activity
therefore strongly suggests that compounds 3, 9 and, by reasonable extension, the other chelators tested
herein primarily exercise their growth inhibition activity through metal chelation and presumably in the
growth medium.
When comparing the susceptibility of the tested microorganisms to each chelator (Table S3), some trends
are evident. Such is the diversity of the cellular architecture, siderophore production capacity, siderophore
identity and activity as well as transport system configurations represented in our panel of
microorganisms, these differences in activity are likely multifactorial and require dedicated mechanistic
study to resolve. In general, C. albicans, S. aureus and B. subtilis are the most sensitive microorganisms
across the range of chelators used. The microorganisms that are least sensitive to these chelators are K.
pneumoniae and E. coli. This trend is largely but not entirely systematic. For example, C. albicans is the
most susceptible tested microorganism to compounds 6, 7, 8 and TACN-MeHP but it is also the least
susceptible microorganism to EMECAM. We have observed the same trends in susceptibility (tested on
the same microorganisms) on a related series of chelators based on 1,2-HOPO containing triaza
macrocyclic cores, suggesting that a dominating factor for efficacy of our molecules is the coordinating
group.40 Also similar to our previous observation is the fact that of the microorganism tested, the Gram-
positive bacteria and the fungus are more susceptible to the tested chelator than the Gram-negative
bacteria. These latter Gram-negative bacteria possess an outer membrane that would provide some
protection against water-soluble agents should they have an intracellular mode of action. This is not
entirely assured as these membranes possess water-filled diffusion channels (porins) that could facilitate
23

our compounds’ diffusion through this extra barrier. Additionally, all of these microorganisms also possess
various drug efflux systems of various specificities. Interestingly, the strain of B. subtilis used is notably
64
limited to the production of a bidentate siderophore ,while both E. coli and K. pneumoniae are known to
produce the very powerful siderophore enterobactin.65-68 The kinetics of iron chelation expected in these
different systems here are consistent with a model based upon extracellular iron chelation.
Hexadentate chelators based on 3,4-HOPO have been studied on their own (i.e. in the absence of added
antimicrobial agents) as in the present study to inhibit bacterial growth.29-31, 35 Contrary to our results, the
authors did not find any significant difference between the susceptibility of Gram-positive and Gram-
negative bacteria to their chelators. Whether the discrepancy between our results and theirs is due to the
strains of microorganisms studied or due to the nature of the chelators remains to be established. The
trend observed across the range of 1,2-HOPO-based chelators described herein and in our previous
study40 and the results of 32 and 33 are all wholly consistent with the hypothesis that metal chelation is
key to biostatic effect. The few outliers in the trends of our MIC values (e.g. high efficacy of DTPA against
S. aureus, compound 9 being one of the most efficacious against S. aureus but one of the poorest
against K. pneumoniae) also suggest that thermodynamically driven Fe3+ chelation is not the only effect
that influences growth of the microorganism. We can speculate that kinetic effects and/or the action of the
chelators on other biologically relevant metals and the susceptibility of the microorganism to withdrawal of
these metals will also play a crucial role that remains to be studied. The coordination chemistry of the 1-
hydroxy-2(1H)-pyridinone coordinating group in hexadentate ligands is still poorly studied. It is therefore
not possible to make definitive judgements on the ability of these chelators to selectively bind Fe3+ in the
2+
2+
2+
presence of other biologically relevant metals such as Cu , Mn , Zn (the amount of relevant metals in
the growth medium was measured and found to be 65.7 M for iron, 31.5 M for zinc, 13.7 M for
manganese, 8.3 M for cobalt and 4.5 M for copper) and therefore how complexation of these
biologically relevant metals could influence growth.
The measured stability constants for 34 and 35 indicate a strong preference of these HOPO derivatives
for Fe3+ over Cu2+ and the higher concentration of iron in the medium suggest that the disruption of iron
homeostasis is however a key factor influencing growth. However, the large difference in MIC between 3
and 9 for K. pneumoniae suggest that the positional isomerism may have a more subtle impact on
24

biological activity than expected from a simple inspection of the pKa and stability constants of the
analogue coordinating groups 34 and 35.
When comparing the general efficacy of each chelator (Table 3), it can be seen that TACN-MeHP, 1, 3
and 6 are almost systematically the most effective. As TACN-MeHP is the most powerful Fe3+ chelator
known, this is not a completely unexpected result. On the contrary, 8 and EMECAM, and to a lesser
extend 9 are almost systematically the least effective compounds. It is interesting that EMECAM, known
as a powerful Fe3+ chelator (pFe3+ = 32.6, compare with enterobactin: pFe3+ = 35.5 and with most natural
siderophores having pFe3+ in the range 25-30 ) is not a powerful biostatic agent against these
microorganisms. This might be associated with some level of siderophore piracy because EMECAM is
based on catechol coordinating groups, widely present in natural siderophores and it is known that
synthetic chelators based on catechol groups can promote bacterial growth.69-70 The addition of a benzo
group on the HOPO (compare 1 and 3) does not significantly alter the chelator’s efficacy, except against
S. aureus. We therefore suggest that the selection of hydroxypyridinone coordinating groups is a shrewd
choice to achieve effective growth inhibition for a diverse range of microorganisms with little chance of
growth promotion. The addition of a benzo group does not appear necessary to prevent siderophore
piracy. It is interesting to note that the difference between the efficacious chelator 3 and the poor chelator
19
8
3
is only on the substitution of the amide nitrogen atom (H and CH respectively). We have not yet
measured the stability constants and pKa of the chelators, hence the effect of the methyl group on the
thermodynamics of metal chelation is not known for these compounds. In the case of catechol-based or
3,2-HOPO-based hexadentate chelators, the addition of a methyl substituent on the nitrogen atom to
remove the hydrogen bonding between the amide’s N-H group and the coordinating atoms of the chelator
did not significantly affect the Fe3+ stability constant of the compound.71-73 The comparison of the efficacy
of 3 (most efficacious) and its isomer 9 (poorer efficacy) indicates that the attachment on the linker onto
the 1,2-HOPO group is also key to obtaining growth inhibition. Once again, this observation may be
rationalised once the pKa and stability constants are measured. This shows that the study of a wide
range of linker groups and an understanding of their effect on metal chelation, including thermodynamic
and kinetic aspects are required to develop chelators for therapeutic applications.
25

Table 3. Ranking of chelator susceptibility (lowest MIC to highest MIC expressed in M) for each microorganism
Microorganism
Chelator Susceptibility
E. coli DSM-18039
TACN-MeHP > 3, 6 > 1, 7, 10 > 9, DTPA > EMECAM > 8
3, TACN-MeHP > 6 > 1, 10 > EMECAM > 7, DTPA > 9 > 8
1, 3, 6 > 7, 10, TACN-MeHP > EMECAM > 9 > DTPA > 8
1 > TACN-MeHP > 3, 6, 7, 9, 10 > 8 > EMECAM > DTPA
TACN-MeHP > DTPA > 3, 6, 9 > 1, 7, 8, 10 > EMECAM
3, 9 > DTPA, TACN-MeHP, 10 > 1, 6 > EMECAM > 7, 8
TACN-MeHP > 7 > 6, 8 > DTPA, 1, 3, 9 > 10 > EMECAM
K. pneumoniae DSM-30104
P. aeruginosa DSM-19880
A. baumannii DSM-30007
B. subtilis DSM-23778
S. aureus DSM-1104
C. albicans DSM-1386
The growth of selected microorganisms with selected chelators over time, at different concentrations of
compounds was followed be measurement of the optical density.
1
.2
E. coli DSM-18039 + 6
A
1
(
)
3
0
0
0
0
.8
.6
.4
.2
0
(
)
4
(
)
2
(1
[
0]:12
[6]:12
[12]:12
[18]:12
) [0]:12
Time (h:min)
26

S. aureus DSM-1104 + 9
1
.4
.2
1
B
1
(3
(4
)
)
0.8
0.6
0.4
0.2
0
(2
)
(1
[
0]:12
[6]:12
[12]:12
[18]:12
)[0]:12
Time (h:min)
1.6
1.2
0.8
0.4
0
C. albicans DSM-1386 + 1
C
(3)
(2)
(1)
[
0]:12
[6]:12
[12]:12
[18]:12
[0]:12
Time (h:min)
Figure 10. Impact of sub MIC concentrations on growth of selected microorganisms. Panel A describes E. coli DSM-
1
8039 grown with 6; Panels B described S. aureus DSM-1104 grown with 9 and panel C describes C. albicans DSM-
386 grown with 1. concentrations: MIC (green, (1)) ; two sub-MIC concentrations MIC / 2 (blue, (2)), and the
1
untreated control (black, (3)).
Each microorganism grew in the absence of chelator with clear lag and an exponential growth phases. In
the presence of the chelators, the lag-phase generally appeared longer and was influenced by the
concentration of chelator, the higher the concentration, the longer the lag-phase. To quantify the impact of
27

the chelator on the lag-phase, we expressed the duration of the lag-phase by the Growth Inhibition Factor
GIF) defined by equation 1:
(
푡푐
퐺퐼퐹 = 푡0 Equation 1
where t
c
0
is the duration of the lag phase in the presence of the chelator and t is the duration of the lag-
phase in the absence of the chelator (Table 4).
Table 4. Calculated Growth Inhibition Factors (GIF) for the apparent length of the lag phase of microbial growth at
sub-MIC ligand concentrations over a period of 24 h. Positive controls for microbial growth were conducted in
quadruplicate and the average reported.
Chelator
7
t0 / min
Concentration
1
3
6
8
9
10
E. coli DSM-18039
MIC / 2
MIC / 4
K. pneumoniae DSM-30104
1.4
1.4
1
1
2.3
1
1.4
1.1
1.3
1.1
1.8
1.4
1.2
1.2
1
1
2
1
2
2
2
80
40
15
95
80
15
75
MIC / 2
MIC / 4
2.4
1.3
1
1
1.9
1
2.3
1.6
+
+
3.6
2
1.4
1.1
P. aeruginosa DSM-19880
MIC / 2
MIC / 4
1.86
1.12
1.12
1.02
1.12
1.12
1.67
1.21
1.4
1.3
2.7
1.95
1.4
1.12
A. baumannii DSM-30007
MIC / 2
MIC / 4
1.64
1.33
1.13
0.82
1.33
1.23
1.44
1.23
2.36
1.74
1.13
0.92
1.95
1.33
B. subtilis DSM-23778
MIC / 2
MIC / 4
1.5
N.R.
1.29
N.R.
1.21
1
1
N.R.
4.5
1.29
1.14
N.R.
A
A
S. aureus DSM-1104
MIC / 2
MIC / 4
0.84
1.02
3.53
1.86
1.49
1.12
1.02
N.R.
2.23
1.12
4.19
3.35
2.98
N.R.
C. albicans DSM-1386
MIC / 2
MIC / 4
3.49
2.47
0.8
X
0.87
0.87
1.02
1.02
0.8
0.87
0.8
0.8
0.87
3.78
(+) Denotes no inhibition of growth was observed at the maximum concentration (5 mM) tested. ‘A’ denotes an
ambiguous result due to ligand precipitation, ‘N.R.’ denotes that no result was taken (and ‘X’ that the sample failed to
grow.
28

As can be seen, the extension of the lag-phase is dependent upon the concentration of chelator applied,
which is wholly consistent with competition for a key nutrient. Until sufficient siderophores (and other
metal acquisition mechanisms, if appropriate) are produced, the growth rate is severely reduced. Once
the metal deprivation effect of the chelator is overwhelmed by the bacteria’s own metal acquisition
system, growth can resume. The higher the concentration of chelator, the longer it takes for the bacteria
to overwhelm the chelator. Some results are, however, puzzling. For example, although C. albicans
appeared quite susceptible to our chelators, the lag-phase generally does not always increase with
chelator concentration. This might indicate a more complex mechanism of action with this fungus; the
chelator could have a biocidal rather than biostatic effect. There does not seem to be any direct
correlation between the GIF value and the MIC (Tables S4-S5). As mentioned before, the microorganisms
that tend to show the lowest MIC value across the tested chelators are C. albicans, S. aureus and B.
subtilis whereas the microorganisms that tend to show the largest GIF are K. pneumoniae, S. aureus.
Similarly, whereas chelators TACN-MeHP, 1, 3 and 6 were almost systematically the ones showing
smallest MIC values, the chelators that show the largest GIF values are 1, 8 and 9 (the GIF value for
TACN-MeHP was not measured).
3- Conclusion
A range of novel secondary/tertiary amine/amide chelators based on 1,2-HOPO and the TREN core has
been prepared and their antimicrobial effects evaluated upon a panel of microorganisms. Our results
suggest that they indeed act as powerful metal chelators and likely inhibit bacterial growth by Fe3+
starvation. It would appear that both the nature of the linker (e.g. amide/amine), the attachment and the
presence of additional substitution on the 1,2-HOPO have a large impact on the MIC values and these
should be taken into consideration when designing therapeutic biostatic chelators. Further work is in
progress to measure the Fe3+ stability constants and pKa of these hexadentate chelators to rationalise
their biological function. Also, more investigations on their mode of action on microorganisms are in
progress to gain a deeper understanding of their ability as biostatic agents against infection.
4- Experimental
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