Probing linker design in citric acid-ciprofloxacin conjugates

Article abstract of DOI:10.1016/j.bmc.2014.04.009

A series of structurally related citric acid-ciprofloxacin conjugates was synthesised to investigate the influence of the linker between citric acid and ciprofloxacin on antibacterial activities. Minimum inhibitory concentrations (MICs) were determined against a panel of reference strains and clinical isolates of bacteria associated with infection in humans and correlated with the DNA gyrase inhibitory activity. The observed trend was rationalised by computational modelling.

Full text of DOI:10.1016/j.bmc.2014.04.009

Accepted Manuscript
Probing linker design in citric acid-ciprofloxacin conjugates
Stephen J. Milner, Anna M. Snelling, Kevin G. Kerr, Ahmad Abd-El-Aziz,
Gavin H. Thomas, Roderick E. Hubbard, Anne Routledge, Anne-Kathrin
Duhme-Klair
PII:
S0968-0896(14)00253-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.04.009
BMC 11504
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
13 February 2014
1 April 2014
6 April 2014
Please cite this article as: Milner, S.J., Snelling, A.M., Kerr, K.G., Abd-El-Aziz, A., Thomas, G.H., Hubbard, R.E.,
Routledge, A., Duhme-Klair, A-K., Probing linker design in citric acid-ciprofloxacin conjugates, Bioorganic &
Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.2014.04.009
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Probing linker design in citric acid-ciprofloxacin conjugates
Stephen J. Milner, Anna M. Snelling, Kevin G.Kerr, Ahmad Abd-El-Aziz, Gavin H. Thomas, Roderick E.
*
*
Hubbard, Anne Routledge , Anne-Kathrin Duhme-Klair .
O
N
O
O
N
O
F
F
OH
OH
N
N
HO H
HO
N
N
N
HOOC
HOOC
HOOC
HOOC
n
O
O
n=0,4
Citrate-functionalized ciprofloxacin conjugates have been synthesized. MICs determined against a panel of clinically relevant
bacterial strains have been determined and correlated with DNA gyrase inhibitory activity. Computational modeling rationalized
observed trend.

Probing linker design in citric acid-ciprofloxacin conjugates
a
c
b,d,e
a,d
d
Stephen J. Milner , Anna M. Snelling , Kevin G. Kerr , Ahmad Abd-El-Aziz , Gavin H. Thomas , Roderick
a,f
a*
a*
E. Hubbard , Anne Routledge , Anne-Kathrin Duhme-Klair
[
a]
5
Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK .
Department of Microbiology, Fewston Wing, Harrogate District Hospital,
[
b]
Harrogate, HG2 7SX, UK.
[
c]
Bradford Infection Group, University of Bradford, Bradford BD7 1DP, UK.
Department of Biology (Area 10), University of York, Heslington, York, YO10 5DD, UK.
Hull York Medical School, Heslington, York, YO10 5DD, UK.
[d]
[
[
e]
f]
1
1
0
5
Vernalis (R&D) Ltd, Granta Park, Cambridge, CB21 6GB, U.K.
Key words: Siderophore, citrate, antibiotic, computational modeling
Abstract: A series of structurally related citric acid-ciprofloxacin conjugates was synthesised to investigate the influence
of the linker between citric acid and ciprofloxacin on antibacterial activities. Minimum inhibitory concentrations (MICs)
were determined against a panel of reference strains and clinical isolates of bacteria associated with infection in humans
2
0 and correlated with the DNA gyrase inhibitory activity. The observed trend was rationalised by computational modeling.
1
. Introduction
‘
Trojan Horse’ antibiotics consisting of a bacterial iron carrier (siderophore) covalently linked to an antimicrobial agent
have attracted much interest due to their potential of overcoming drug-access related resistance, in particular permeability
1
-4
2
3
5 barrier and efflux-related mechanisms.
Most of the siderophore-drug conjugates investigated to date use β-lactam antibiotics as the drug component, mainly
because of their relatively accessible drug targets. More recently, fluoroquinolone antimicrobials, which act through
inhibition of cytoplasmic DNA gyrase or topoisomerase IV, have also been explored. Some of the fluoroquinolone
conjugates were found to retain antimicrobial activity, thereby demonstrating that the drug components can reach their
5
-8
9,10
11,12
0 intracellular drug targets. Thus far fluoroquinolone-conjugates with hydroxamate-, catecholate-
and citrate-type

1
3
14,15
siderophores and their mimics, pyoverdin and pyochelin
have been reported. Interestingly, it was found that the
properties of the linker between the siderophore and the fluoroquinolone can be crucial in determining the antibacterial
5
,9,13,14
activity of the conjugates.
Previously, we investigated two regioisomeric citrate-ciprofloxacin conjugates, 1 and 2. Citrate and citrate-type
5
siderophores are used by many bacteria to mediate iron(III) uptake. Whilst the role of citrate as exogenous siderophore is
1
6-19
20
best understood in Escherichia coli,
a number of other Gram-negative and Gram-positive bacteria are known to
import ferric citrate. 1 and 2 were found to retain antibacterial activity against ciprofloxacin-susceptible bacterial strains;
11
however, both conjugates were less potent than the parent drug ciprofloxacin.
To improve the antimicrobial activity of the conjugates, the linker that connects the citric acid unit with the piperazinyl
1
0 nitrogen of ciprofloxacin required optimisation. Herein we report the synthesis and antimicrobial activities of a series of
structurally related conjugates designed to probe the effect of the length of the linker.
O
O
F
OH
1
1,3-c-cpf
N
N
HO
HOOC
HOOC
N
O
O
O
F
OH
HOOC
HOOC
N
N
2 1,5-c-cpf
N
HO
O
O
N
O
F
OH
OH
3
4
1,3-c-Gly-cpf
1,3-c-Ava-cpf
HOOC
O
N
HOOC
N
N
HO
H
O
O
N
O
F
N
H
HO
N
N
HOOC
HOOC
O
O
Fig 1 Citric acid-ciprofloxacin conjugates
2
. Results and Discussion
1
2
5 Design and synthesis of the conjugates
Within a series based on 1, glycine- and aminovaleric acid were introduced as spacers between the citryl-moiety and
ciprofloxacin to give conjugates 3 and 4, respectively. These represent partial structures that can be derived from the
12
ciprofloxacin conjugate of the natural citrate-based siderophore, staphyloferrin A, as shown in Scheme 1. In conjugates 3
0 and 4 the citryl-unit is linked through one of the primary carboxyl groups. As a consequence, the quaternary citryl-carbon
in the resulting products is chiral. Since the configuration of the citryl-moieties in staphyloferrin A is still under
21-23
discussion,
3 and 4 were synthesised as racemic mixtures and screened as such.

O
N
O
F
OH
N
O
N
H
O
N
NH
HOOC
O
OH
COOH HOOC
OH
COOH
O
N
O
O
O
F
F
OH
OH
N
N
N
O
N
O
N
H
O
N
NH
HOOC
O
OH
COOH
OH
4
COOH
3
HOOC
Scheme 1 Citric acid-ciprofloxacin conjugates derived from the ciprofloxacin staphyloferrin A conjugate
O
O
N
COOR1
F
COOR1
(iii)
F
N
N
N
H N
OH
BocHN
OH
BocHN
N
H N
N
2
3
n
O
n
O
n
O
n
(ii)
(i)
O
n = 0, n = 4
5a n = 0
n = 4
(iv)
5
b n = 4
O
N
F
COOR1
R1 = Me
N
OH
H
N
N
1
R OOC
n
1
(v)
R OOC
O
O
6
6
a n = 0
b n = 4
R1 = Na+
, 4
3
5
Scheme 2 Reagents and conditions: (i) (Boc)
71%. (iii) 4 M HCl in MeOH, 0 ºC-r.t. 1 h. (iv) 1,3-dimethylcitrate, EDC.HCl, HOBt.H
h, 3 =68%, 4 =50%.
2
O, NaHCO
3
, H
2
O/1,4-dioxane, r.t., 24 h, 65%. (ii) EDC.HCl, HOBt.H
2
O, DMF, r.t. 24 h, 5a =65%, 5b
=
2
O, DMF, r.t. 5 d, 6a =72%, 6b =75% (v) TBAH in MeOH, r.t. 18

11
The synthetic strategy for 3 and 4 is shown in Scheme 2, 1 was prepared as previously reported. Boc-protected spacer
units were reacted with the methyl ester of ciprofloxacin using an EDC-mediated coupling to give 5a,b in good yields.
Subsequent Boc-deprotection and reaction with 1,3-dimethyl citrate gave the methyl-protected precursors 6a,b. Ester
hydrolysis furnished conjugates 3 and 4.
5
Antibacterial activity
To assess the influence of the citric acid moiety and the spacers on the antimicrobial activity of the parent drug, the
conjugates were screened against a collection of reference and clinical isolates of bacteria associated with infection in
humans (for details see Table 1) to determine the minimum inhibitory concentration (MIC). All conjugates showed a
1
0 significantly lower activity than ciprofloxacin on its own. Whilst conjugates 1 and 3 showed similar activities against
Gram-positive strains, the introduction of the glycine spacer in 3 led to slightly improved activity against five of the seven
tested ciprofloxacin-susceptible Gram-negative strains. On the other hand, the introduction of aminovaleric acid as spacer
(
conjugate 4) gave rise to a significant reduction in potency across the panel of strains tested.
Gyrase assay
1
2
2
5 Next we were interested in investigating if the observed decrease in antimicrobial activity upon conjugate formation
correlates with a decrease in affinity for the drug target. To assess how the citric acid unit and the linker structure affects
the ability of the antimicrobial to inhibit the activity of DNA gyrase, we measured DNA gyrase inhibitory activity of 1, 3
and 4 using a commercial DNA gyrase supercoiling assay (Inspiralis). In this semi-quantitative assay a non-supercoiled
template plasmid is supercoiled through the action of the DNA gyrase which results in a detectable mobility shift of the
0 plasmid DNA when electrophoresed on an agarose gel (Figure 2). Using free ciprofloxacin, complete inhibition of DNA
gyrase activity can be observed at concentrations over 5 μM, where no supercoiled form can be observed. For conjugates 1,
3
and 4 there is also clear inhibition of gyrase activity, but small amounts of supercoiled forms can still observed even with
0 μM compound, suggesting that these compounds are less effective at inhibiting DNA gyrase than the free drug. Of the
2
three conjugates, compound 1 appears to be the most effective, being only marginally less inhibitory, perhaps 2-fold, than
5 the free drug, whereas compounds 3 and 4 show significantly reduced activity. This drop in gyrase inhibitory activity is
consistent with the observed overall decrease in antimicrobial potency seen against Gram-negative bacterial strains. A
similar observation was reported by Abdallah et al. for two pyoverdin-norfloxacin conjugates, which were approximately
13
5
0-times less active in inhibiting DNA gyrase than norfloxacin alone. As the spacer length increases, the DNA gyrase

inhibitory effect decreases. We had hoped that the introduction of a longer spacer would alleviate steric hindrance or
electrostatic repulsion exerted by the citryl-unit and thus increase the inhibitory activity. However, this was not observed.
Instead, the insertion of an aminovaleric acid spacer led to clear decrease in both gyrase inhibitory and antimicrobial
activity. The strong correlation between gyrase activity and antimicrobial activity suggests that this may be the main factor
responsible for the loss in antimicrobial potency. If this is the case it suggests that modifications in linker structure have not
altered how the conjugate moves into the cell, which is likely through the OmpF porin in the outer membrane and possibly
diffusion through the cytoplasmic membrane.
5
Fig. 2. Ethidium bromide-stained agarose gel showing data from DNA gyrase assays (OC: open complex form; SC: supercoiled form; * indicates no
1
0
sample loaded).
1
2
2
3
5
0
5
0

Table 1: Activity of citrate-ciprofloxacin conjugates 1, 3 and 4.
control
conjugates
ciprofloxacin
1
3
4
bacterial strains
MIC
MIC
µg/
mL
16
R
MIC
MIC
a
a
a
µg/
mL
nmol/
mL
nmol/ ratio
mL
µg/
mL
nmol/ ratio
µg/
mL
nmol/
mL
ratio
mL
28.4
R
Staphylococcus aureus (Oxford)
NCTC 6571
Staphylococcus aureus (HG-1)
Staphylococcus aureus-15 NCTC
0
.25 0.755
31.6
41.8
16
R
37.6
R
R
R
R
R
R
c d
b
R
R
e
0.5
1.51
8
15.8
10.5
16
28.4
18.8
1
3142
Staphylococcus aureus-16 NCTC
c e
R
R
R
R
R
R
R
R
R
R
R
1
3143
Staphylococcus aureus BIG 0052
Staphylococcus epidermidis NCTC
1047
Staphylococcus epidermidis NCTC
749
Staphylococcus haemolyticus NCTC
1042
c d
0.5
1.51
16
16
31.6
31.6
20.9
83.8
16
16
28.4
28.4
18.8
75.3
0
.125 0.377
1
0
.25 0.755
8
15.8
20.9
8
14.2
28.4
18.8
32
R
52.9
R
70
8.8
35
2
0
.125 0.377
16
31.6
0.5
83.8
2.8
16
75.3
1.23
1
Escherichia coli NCTC 10418
0.06 0.181
R
R
0.25
R
R
0.125 0.222
R
R
1
R
R
1.6
R
R
c
Escherichia coli BIG 0046
Coliform BIG 0051
R
R
R
R
c
Pseudomonas aeruginosa
0
.25 0.755
6.04
.25 0.755
4
7.9
10.5
5.2
2
3.56
28.4
4.72
4.7
16
R
26.5
R
(Environmental) BIG 0039
Pseudomonas aeruginosa (Clinical)
BIG 0037
Pseudomonas aeruginosa NCTC
2
16
31.6
16
0
4
4
1
7.9
7.9
2
10.5
10.5
11
2
8
3.56
14.2
4.72
18.8
4.91
16
16
4
26.5
26.5
6.6
35
35
1
0662
Pseudomonas aeruginosa BIG 0063
Serratia marcescens BIG 0011 =
NCTC 1377
Burkholderia cepacia BIG 0009 =
NCTC 10744
Burkholderia cepacia BIG 117
Burkholderia cepacia BIG 118
Burkholderia cepacia BIG 119
Burkholderia cepacia BIG 120
Burkholderia cepacia BIG 121
Stenotrophomonas maltophilia
N1127
0.25 0.755
.06 0.181
0
0.5
0.889
36.5
35
0
.5
1.51
3.02
16
31.6
20.9
8
14.2
9.40
32
52.9
1
16
16
R
16
R
31.6
31.6
R
31.6
R
10.5
41.8
8
8
32
32
R
14.2
14.2
56.9
56.9
R
4.70
18.8
4.7
R
R
R
R
R
R
R
R
R
R
0.25 0.755
4
2
8
12.1
6.04
24.1
5.2
9.4
2
4
1
6.04
12.1
3.02
32
R
63.3
R
10.5
32
32
R
56.9
56.9
R
9.4
4.7
R
R
R
R
R
R
Stenotrophomonas maltophilia
N1124
Stenotrophomonas maltophilia
N1119
32
63.3
20.1
a
ratio = MIC of conjugate (nmol/mL) / MIC of ciprofloxacin (nmol/mL)
R = resistant, MIC > 36 μg/mL.
ciprofloxacin resistant strain
methicillin-resistant Staphylococcus aureus (MRSA)
epidemic methicillin-resistant Staphylococcus aureus (EMRSA)
b
c
d
e
5

Molecular modeling
The crystal structure of a tertiary complex between a Staphylococus aureus DNA gyrase construct, ciprofloxacin and
24
cognate DNA has previously been determined (pdb code: 2xct). Figure 3 shows how in this structure, ciprofloxacin
intercalates between T:A and G:C pairs to stall replication. As shown in Figure 3, the carboxyl moiety of ciprofloxacin
makes specific interactions with S1084 and S1085 at the N terminus of a helix in one of the gyrase monomers and although
there are incidental interactions with the same monomer along the piperazine moiety, the primary vector exploited in the
analogues points into the solvent.
5
There is 43.2% identity overall between the sequences of S. aureus and E. coli gyrase, and 67% similarity in the amino
acids within 5 Å of ciprofloxacin. It is therefore reasonable to use the S. aureus structure as a model for the E. coli enzyme.
1
1
2
0 Models were constructed for the binding of conjugates 1, 3 and 4 to the gyrase/DNA complex and an energy of interaction
determined (Table 2). There is a glutamic acid within about 4.5 Å of the secondary amine of the piperazine ring of
ciprofloxacin; this glutamic acid is in a salt bridge with an arginine. There will therefore be little impact on recognition by
acylation of the piperazine in the derivatives, as reflected in the calculation. Although these models are relatively
unsophisticated, the general trend in energy reflects the measured inhibitory activity, with longer linkers having a weaker
5 energy of interaction in the model. The models were constructed using the S. aureus structure, but as emphasised in Figure
4
, there is strong similarity of the amino acids which make the major interactions in the channel into which the conjugates
bind in the model. These calculations suggest that a long linker group provides less stable binding to the DNA gyrase and
that the optimal linker group should not contain more than 1-2 atoms. The major differences between S. aureus (which was
used to make the model) and E. coli gyrase (which provided the experimental data) are in the amino acids which
0 accommodate the longer linker (Gln489->Thr and Arg479->Ala). These changes would only increase the destabilisation of
the binding of the longer linker conjugates in our model.
Fig. 3 Detail of the ciprofloxacin/DNA/gyrase structure. Protein backbone is in grey ribbon, DNA is with yellow arrow backbone and nucleic acids
2
5
represented as coloured rods, with ciprofloxacin in ball and stick representation coloured by element.

Fig. 4 Schematic of the interactions made by the computationally determined pose of 1,3-c-Ava-cpf, 4, in the S. aureus DNA gyrase complex
corresponding E. coli residues in parentheses). The ciprofloxacin core for the different conjugates varied by less than 0.8 Å from the original in the 2xct
(
5
structure, and the same interactions were made by each conjugate.
Table 2: Computationally determined energies of ciprofloxacin derivatives.
Compound
Potential energy
-
1
(
kcal/mol )
-2385
Ciprofloxacin
1
,3-c-cpf, 1
,3-c-Gly-cpf, 3
,3-c-Ava-cpf, 4
-2273
-2218
-1751
1
1
1
0
3
. Conclusions
A series of structurally related ciprofloxacin conjugates was synthesised and screened against a panel of bacteria associated
with infection in humans. Within the series, the DNA gyrase inhibitory potency decreases with increasing spacer length and
the observed effect correlates with the decrease in antimicrobial activity against Gram-negative bacterial strains. Hence, the
1
5 insertion of the glycine- and aminovaleric acid spacers does not facilitate the binding to and the inhibition of the drug
target. In particular, the insertion of the aminovaleric acid spacer caused a significant decrease in both gyrase inhibitory and
antimicrobial activity. Molecular modeling studies confirmed that the long linker group provides less stable binding to
DNA gyrase and the calculations suggest that a linker should not contain more than 1-2 atoms.

Future studies will be directed towards the enhancement of the DNA inhibitory and antimicrobial activities of Trojan horse
prodrugs aimed at exploiting active transport mechanisms, but based on biolabile linkers designed to release ciprofloxacin
from the delivery unit once inside the cell.
4
. Experimental section
5
4.1 General
Chemical reagents were purchased from Aldrich or Fluka and were used as supplied. 1,3– and 1,5-dimethyl citrate and the
11,12
methyl esters of ciprofloxacin were prepared as described previously.
Solvents were supplied by Aldrich or Fisher
1
13
Scientific and used as received or dried over 3 or 4 Å molecular sieves prior to use. H NMR (399.785 MHz), C NMR
1
9
(
100.535 MHz) and F NMR (376.152 MHz) spectra were acquired on a Jeol ECX400 or ECS400 spectrometer in the
1
1
2
2
0 stated deuterated solvents. Chemical shifts (δ) are reported in parts per million (ppm) using the solvent residual peak as an
1
13
internal standard for H and C NMR spectra: 7.26 ppm and 77.2 ppm respectively for CDCl ; 2.50 ppm and 39.5 ppm
3
respectively for (D C) SO; 3.31 ppm and 49.0 ppm respectively for CD OD. Spectra performed in D O were spiked with
3
2
3
2
13
CD CN and its solvent residual peaks used as an internal standard for C NMR: 1.32/118.3 ppm. Fluorobenzene was used
3
1
9
as an internal standard for F NMR spectra, taken as -113.150 ppm with reference to CFCl as 0 ppm. Coupling constants
3
1
3
5 (J) are reported in Hertz (Hz). All C experiments were confirmed using DEPT experiments. Electrospray ionisation (ESI)
mass spectra were acquired on a Thermo-Finnigan LCQ mass spectrometer. Infrared spectra were recorded on a Thermo
Nicolet Avatar FTIR spectrometer as KBr discs or as a solution in the stated anhydrous solvent. Melting points were
acquired on a Stuart Scientific SMP3 apparatus and are uncorrected.
0 4.2 DNA gyrase assay
Assays were performed using the DNA gyrase supercoiling assay from Inspiralis according to the manufacturer’s
25
instructions and based on the method of Maxwell et al. Success of the assay was absolutely dependent on the use of a gel
electrophoresis apparatus that had never come into contact with ethidium bromide as clearly extremely small amounts of
this compound can lead to total failure of the assay to resolve the different super-coiled forms of the plasmid.
5
4
.3 Computational modelling
26
27
All modelling was performed with Accelrys Discovery Studio 3.0 and CHARMm . The models were constructed from
the Staphylococcus aureus/DNA gyrase/ciprofloxacin complex structure (pdb: 2xct). The citrate derivatives were

constructed by editing the initial ciprofloxacin. Appropriate hydrogen atoms and formal charges were then added and the
27
structure was typed using CHARMm forcefields and Momany-Rone partial charges. The structure was then minimized
using fixed atom constraints for all protein and DNA atoms using the Smart Minimizer algorithm of Discovery Studio 3.0
with a maximum of 200 steps. The stability of these models was assessed by performing molecular dynamics simulations to
reach equilibrium within 1000 steps of 0.001 s at a target temperature of 300 K with no fixed atom positions, followed by
minimisation. The final structures derived for each complex were within an RMSD of <1.0 Å, with only minor changes
arising from the molecular dynamics. One low energy pose was seen for each of the conjugates except for 1,3-c-Ava-cpf
which has two equally low energy conformations, probably due to the length of the aminovaleric acid linker group
introducing flexibility into the conjugated ligand.
5
1
1
2
2
0 4.4 Screening
Bacterial strains were maintained on Iso-Sensitest Agar (Oxoid). The Minimum Inhibitory Concentration (MIC) of
ciprofloxacin and the conjugates was determined using micro dilution in 96 well microtitre plates (Sterilin) in accordance
with BSAC susceptibility test guidlines. Strains were exposed to doubling concentrations of the chemicals in Iso-Sensitest
o
broth (Oxoid), with incubation for 18h at 37 C, before the lowest concentration that inhibited growth (as determined by
5 lack of turbidity) was recorded.
4
.5 Synthesis
Methyl-7-[4-(2-{[(tert-butoxy)carbonyl]amiacetyl)piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylate (5a)
To a stirred solution of methyl 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinolone-3-carboxylate (0.23
0 g, 0.64 mmol), Boc-Gly-OH (0.11 g, 0.64 mmol) and HOBt.H O (0.09 g, 0.64 mmol) in dry DMF (18 mL) were added
2
DIPEA (115 μL, 0.66 mmol) and EDC.HCl (0.12 g, 0.64 mmol). The resultant mixture was left to stir at rt. for 24 h and the
solvent removed in vacuo. The residue was diluted with deionised water (60 mL) and then extracted with DCM (3 × 80
mL). The organic extract was then washed with water (50 mL), aqueous hydrochloric acid (50 mL, 0.05 M), aqueous
sodium hydrogen carbonate (50 mL, 0.05 M), brine (50 mL) and water (50 mL). The organic extract was dried over
5 MgSO , filtered and dried in vacuo to afford a white residue. The residue was precipitated with DCM:diethyl ether, filtered
4
-
1
and washed with diethyl ether to afford 220 mg 5a (65%) as a white powder. IR (KBr, cm ) 3424br (NH), 3299br (NH),
2
1
1
976w (CH), 2951w (CH), 1717s (C=O), 1704s (C=O), 1655s (C=O), 1623s (C=O), 1589m, 1558w, 1545m, 1495s (C-O),
477s (C-O), 1456s (C-O), 1440s (C-O), 1391m, 1366m, 1350m, 1328m, 1316w, 1283w, 1242m, 1212w, 1166m, 1107w,
1
3
4
089w, 1055w, 1027m. H NMR (CDCl , 400 MHz) δ 8.51 (1H, s), 7.98 (1H, d, J = 13.1 Hz), 7.24 (1H, d, J = 7.0
3
H
H-F
H-F

3
Hz), 5.50 (1H, br s, H), 4.03 (2H, d, J = 4.4 Hz), 3.89 (3H, s), 3.86-3.84 (2H, m), 3.62-3.60 (2H, m), 3.45-3.39 (1H, m),
H-H
13
3
.25-3.21 (4H, m), 1.44 (9H, s), 1.34-1.29 (2H, m), 1.14-1.11 (2H, m). C NMR (CDCl , 100 MHz) δ 173.0 (C=O), 167.1
3 C
1
2
(
C=O), 166.3 (C=O), 155.9 (C=O), 153.4 (ipso-Ar, d, J = 249 Hz), 148.6 (CH), 144.0 (ipso-Ar, d, J = 10.7 Hz),
C-F C-F
3
2
3
1
38.0 (ipso-Ar), 123.7 (ipso-Ar, d, J = 6.9 Hz), 113.5 (CH, d, J = 23.0 Hz), 110.2 (ispo-Ar), 105.4 (CH, d, J
=
C-F
C-F
C-F
5
3.08 Hz), 80.0 (4º-C), 52.2 (CH ), 50.15 (CH ), 49.7 (CH ), 44.4 (CH ), 42.3 (CH ), 41.9 (CH ), 34.7 (CH), 28.4 (CH x3),
3 2 2 2 2 2 3
+
+
8
.3 (CH x2). m/z (ESI); 503 ([M+H] , 100 %); HRMS (ESI) Calc. for C H N O F 503.2300. Found [M+H] , 503.2312
2 25 32 4 6
(
2.0 ppm error).
1
,4-Dimethyl-2-{[(2-{4-[1-cyclopropyl-6-fluoro-3-(methoxycarbonyl)-4-oxo-1,4-dihydroquinolin-7-yl]piperazin-1-yl}h-2-
1
1
2
2
0 oxoethyl)carbamoyl]methyl}-2-hydroxybutanedioate (6a)
To stirred solution of ice-cold HCl in MeOH (20 mL) was added methyl 7-[4-(2-{[(tert-
a
4 M
butoxy)carbonyl]amino}acetyl)piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate 5a (0.184
g, 0.366 mmol) and the mixture left to stir at rt. for 16 h. The solvent was removed and the resultant yellow residue dried
overnight in a vacuum oven.
5 The yellow residue was dissolved in dry DMF (15 mL), DIPEA (0.27 mL, 1.54 mmol) and 4 Å molecular sieves (~0.5 g)
added. After stirring at rt. for 30 mins, 3-hydroxy-5-methoxy-3-(methoxycarbonyl)-5-oxopentanoic acid (0.068 g, 0.308
mmol), HOBt.H O (0.042 g, 0.308 mmol) and EDC.HCl (0.059 g, 0.308 mmol) were added. The resultant mixture was left
2
to stir at rt. for 5 d, filtered, washed with CHCl (10 mL) and the solvent removed in vacuo.
3
The residue was diluted with water (60 mL) and then extracted with DCM (3 × 80 mL). The organic extract was then dried
0 over MgSO , filtered and the solvent removed in vacuo. The residue was redissolved in DCM (80 mL) and washed with
4
0
.05 M MeSO H (20 mL), H O (20 mL), 0.05 M NaHCO (20 mL), brine (20 mL) and H O (20 mL). The organic extract
3 2 3 2
was dried over MgSO , filtered and dried in vacuo to afford a pale yellow solid. The product was triturated in diethyl ether,
4
-
1
filtered and washed with diethyl ether to afford 150 mg 6a (72%) as a off white powder. IR (KBr, cm ) 3407br (OH),
3
096w (CH), 3005w (CH), 2952w (CH), 2850w (CH), 1723s (C=O), 1620s (C=O), 1588m, 1547m, 1493s, 1478s, 1440s,
1
5 1388m, 1348m, 1316m, 1283m, 1243s (C-O), 1210s (C-O), 1166m (C-O), 1104m, 1088m, 1026m. H NMR (CDCl , 400
3
3
4
3
MHz) δ 8.54 (1H, s), 8.05 (1H, d, J = 13.1 Hz), 7.28 (1H, d, J = 5.9 Hz), 7.04 (1H, br t, J = 3.9 Hz), 4.66 (1H,
H
H-F
H-F
H-H
s), 4.13-4.12 (2H, m), 3.91 (3H, s), 3.86-3.83 (1H, br m), 3.82 (3H, s), 3.75-3.72 (1H, m), 3.69 (3H, s), 3.63-3.61 (2H, br
13
m), 3.46-3.40 (1H, m, H-3), 3.34-3.22 (4H, br m), 3.07-2.70 (4H, m), 1.35-1.32 (2H, m), 1.14-1.12 (2H, m). C NMR
CDCl , 100 MHz) δ 174.1 (C=O), 173.1 (C=O), 170.6 (C=O), 169.4 (C=O), 166.5 (C=O), 166.4 (C=O), 153.5 (ipso-Ar,
(
3
C

1
2
3
d, J = 248 Hz), 148.7 (CH), 144.0 (ipso-Ar, d, J = 10.7 Hz), 138.1 (ipso-Ar), 124.0 (ipso-Ar, d, J = 6.9 Hz), 113.8
C-F
C-F
C-F
2
(
(
CH, d, J = 22.2 Hz), 110.4 (ipso-Ar), 105.4 (CH), 73.9 (4º-C), 53.3 (CH ), 52.3 (CH ), 52.2 (CH ), 50.2 (CH ), 49.8
C-F 3 3 3 2
19
CH ), 44.5 (CH ), 44.4 (CH ), 43.0 (CH ), 42.0 (CH ), 41.4 (CH ), 34.7 (CH), 8.3 (CH x2). F NMR (CDCl , 376 MHz)
2
2
2
2
2
2
2
3
+
δF -124.03 (dd, JH-F = 12.6, 6.9 Hz). m/z (ESI); 605 ([M+H] , 100 %); HRMS (ESI) Calc. for C H N O F 605.2253.
28
34
4
10
+
5
Found [M+H] , 605.2273 (2.9 ppm error).
2
-[({2-[4-(3-Carboxy-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)piperazin-1-yl]-2-
oxoethyl}carbamoyl)methyl]-2-hydroxybutanedioic acid (3)
To a solution of 0.1 M Bu NOH (3.3 mL, 0.331 mmol; made by dilution of 1 M TBAH in MeOH with H O) was added
4
2
1
1
2
2
0 1,4-dimethyl 2-{[(2-{4-[1-cyclopropyl-6-fluoro-3-(methoxycarbonyl)-4-oxo-1,4-dihydroquinolin-7-yl]piperazin-1-yl}6-2-
oxoethyl)carbamoyl]methyl}-2-hydroxybutanedioate 6a (0.05 g, 0.0827 mmol) and the mixture stirred at rt. for 18 h.
The pH of the solution was reduced to pH 3.5 through addition of 0.2 M pyruvic acid (~1.8 mL). The resultant precipitate
was filtered, washed with ice-cold H O (2.5 mL), 1,4-dioxane (5 mL) and CHCl (75 mL) and dried on the vacuum line to
2
3
-
1
afford 32 mg of 3 (68%) as an off white powder. IR (KBr, cm ) 3417br (OH), 3061w (CH), 2928w (CH), 1722m (C=O),
5 1628s (C=O), 1544w, 1509m (C-O), 1493m, 1469m (C-O), 1390w, 1335w, 1268w, 1246m, 1214w, 1189w, 1149w, 1131w,
1
3
1
113w, 1026w. H NMR (d -DMSO, 400 MHz) δ 15.20 (1H, s), 8.67 (1H, s), 8.10 (1H, br t, J = 5.2 Hz), 7.94 (1H, d,
6 H H-H
3
4
3
JH-F = 13.1 Hz), 7.59 (1H, d, J = 7.3 Hz), 4.03 (2H, d, J = 5.2 Hz), 3.83-3.82 (1H, m), 3.69-3.66 (4H, br m), 3.36
H-F
H-H
1
3
(
(
4H, br s), 2.76-2.54 (4H, m), 1.33-1.31 (2H, m), 1.19 (2H, br s). C NMR (d -DMSO, 100 MHz) δ 176.4 (C=O), 175.1
6 C
1
C=O), 171.4 (C=O), 169.5 (C=O), 167.0 (C=O), 165.9 (C=O), 153.0 (ipso-Ar, d, J = 249 Hz), 148.1 (CH), 144.9 (ipso-
C-F
2
3
3
4
0 Ar, d, J = 10.7 Hz), 139.1 (ipso-Ar), 118.9 (ipso-Ar, d, J = 7.7 Hz), 111.0 (CH, d, J = 23.0 Hz), 106.7 (CH, J
C-F
C-F
C-F
C-F
19
=
7.7 Hz), 72.7 (4º-C), 49.4 (CH ), 49.2 (CH ), 43.8 (CH ), 43.3 (CH ), 41.0 (CH ), 40.6 (CH ), 35.9 (CH), 7.6 (CH x2). F
2 2 2 2 2 2 2
NMR (d -DMSO, 376 MHz) δ -121.90 (dd, J = 19.0, 11.5 Hz).
6
F
H-F
+
+
m/z (ESI); 563 ([M+H] , <1 %); HRMS (ESI) Calc. for C H N O F 563.1784. Found [M+H] , 563.1778 (1.0 ppm error).
25
28
4
10
5 5-{[(tert-Butoxy)carbonyl]amino)pentanoic acid
To a stirred solution of 5-aminovaleric acid (1.5 g, 12.8 mmol) and sodium hydrogen carbonate (2.69 g, 32.0 mmol) in
deionised water (25 mL) was added di-tert-butyl dicarbonate (3.35 g, 15.4 mmol) in 1,4-dioxane (25 mL) and left to stir at
rt. for 24 h. The resultant mixture was filtered and the filtrate washed with diethyl ether (25 ml). The aqueous layer was
reduced to pH 2 through the addition of aqueous hydrochloric acid (~7 mL, 2 M) and then extracted with ethyl acetate (3 ×

1
5 mL). The organic extract was dried over MgSO , the solvent removed in vacuo and the resultant residue precipitated
4
-
1
(
(
diethyl ether/n-hexane) to afford 1.80g of the title compound (65%) as a white crystalline powder. IR (KBr, cm ) 3374s
OH), 3021m (CH), 3003m (CH), 2984m (CH), 2971m (CH), 2952m (CH), 2877m (CH), 1713s (C=O), 1686s (C=O),
1
653w, 1524s (C-O), 1486w, 1462w, 1445w, 1430w, 1415w, 1391w, 1364m, 1333w, 1279s, 1245s, 1206s, 1170s, 1136w,
1
3
5
1058w. H NMR (CDCl , 400 MHz) δ 10.96 (1H, br s), 4.63 (2H, br s), 3.14-3.11 (2H, br m), 2.36 (2H, t, J = 7.3 Hz),
3 H H-H
13
1
.69-1.61 (2H, m), 1.58-1.49 (2H, m), 1.43 (9H, s). C NMR (CDCl , 100 MHz) δ 178.8 (C=O), 156.2 (C=O), 79.4 (4º-
3 C
+
C), 40.2 (CH ), 33.7 (CH ), 29.5 (CH ), 28.5 (CH x3), 22.0 (CH ). m/z (ESI); 240 ([M+Na] , 100 %); HRMS (ESI) Calc.
2
2
2
3
2
+
for C H NO Na 240.1206. Found [M+Na] , 240.1201 (2.1 ppm error).
10
19
4
1
1
2
2
0 Methyl-7-[4-(5-{[(tert-butoxy)carbonyl]amino}pentanoyl)piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylate (5b)
To a stirred solution of methyl 1-cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)-1,4-dihydroquinolone-3-carboxylate (0.23
g, 0.64 mmol), 5-{[(tert-butoxy)carbonyl]amino)pentanoic acid (0.14 g, 0.64 mmol) and HOBt.H O (0.09 g, 0.64 mmol) in
2
dry DMF (18 mL) were added DIPEA (115 μL, 0.66 mmol) and EDC.HCl (0.12 g, 0.64 mmol). The resultant mixture was
5 left to stir at rt. for 24 h and the solvent removed in vacuo. The residue was diluted with deionised water (60 mL) and then
extracted with DCM (3 × 80 mL). The organic extract was then washed with deionised water (50 mL), aqueous
hydrochloric acid (50 mL, 0.05 M), aqueous sodium hydrogen carbonate (50 mL, 0.05 M), brine (50 mL) and deionised
water (50 mL). The organic extract was dried over MgSO , filtered and dried in vacuo to afford a pale yellow residue. The
4
residue was precipitated with DCM:diethyl ether, filtered and washed with diethyl ether to afford 260 mg of 5b (71%) as a
0 pale yellow powder.
-
1
IR (KBr, cm ) 3291br (NH), 3073w (CH), 2966w (CH), 2947w (CH), 2868w (CH), 1737s (C=O), 1700s (C=O), 1635s,
1
1
1
622s, 1591w, 1549w, 1492m (C-O), 1474m (C-O), 1457w, 1441m, 1388w, 1364w, 1349w, 1330w, 1317w, 1265m, 1247m,
1
3
209w, 1171m, 1158m, 1093w, 1035w, 1028w, 1001w. H NMR (CDCl , 400 MHz) δ 8.51 (1H, s), 7.98 (1H, d, J =
H-F
3
H
4
3.1 Hz), 7.24 (1H, d, J = 7.0 Hz), 4.67 (1H, br s), 3.88 (3H, s), 3.84-3.81 (2H, m), 3.69-3.67 (2H, m), 3.45-3.39 (1H,
H-F
3
5 m), 3.25-3.23 (2H, m), 3.20-3.18 (2H, m), 3.16-3.11 (2H, m), 2.40 (2H, t, J
= 7.4 Hz), 1.73-1.65 (2H, m), 1.58-1.51
H-H
13
(
2H, m), 1.41 (9H, s), 1.34-1.29 (2H, m), 1.15-1.11 (2H, m). C NMR (CDCl , 100 MHz) δ 173.1 (C=O), 171.5 (C=O),
3
C
1
2
1
66.4 (C=O), 156.2 (C=O), 153.4 (ipso-Ar, d, JC-F = 249 Hz), 148.6 (CH), 144.2 (ipso-Ar, d, J = 10.7 Hz), 138.0 (ipso-
C-F
3
2
3
Ar), 123.6 (ipso-Ar, d, J = 6.9 Hz), 113.5 (CH, d, J = 23.0 Hz), 110.2 (ipso-Ar), 105.3 (CH, d, J = 2.3 Hz), 79.2
C-F
C-F
C-F
(
4º-C), 52.2 (CH ), 50.6 (CH ), 49.8 (CH ), 45.5 (CH ), 41.4 (CH ), 40.1 (CH ), 34.7 (CH), 32.7 (CH ), 29.8 (CH ), 28.5
3 2 2 2 2 2 2 2

1
9
(
(
CH x3), 22.3 (CH ), 8.3 (CH x2). F NMR (CDCl , 376 MHz) δ -123.92 (dd, J = 12.6, 6.9 Hz). m/z (ESI); 545
3 2 2 3 F H-F
+
+
[M+H] , 100 %); HRMS (ESI) Calc. for C H N O F 545.2770. Found [M+H] , 545.2791 (3.8 ppm error).
2
8
38
4
6
1
,4-Dimethyl-2-{[(5-{4-[1-cyclopropyl-6-fluoro-3-(methoxycarbonyl)-4-oxo-1,4-dihydroquinolin-7-yl]piperazin-1-yl}-5-
5
oxopentyl)carbamoyl]methyl}-2-hydroxybutanedioate (6b)
To stirred solution of ice-cold HCl in MeOH (20 mL) was added methyl 7-[4-(5-{[(tert-
butoxy)carbonyl]amino}pentanoyl)piperazin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate 5b
0.20 g, 0.367 mmol) and the mixture left to stir at rt. for 16 h. The solvent was removed in vacuo and the resultant yellow
residue dried overnight in a vacuum oven.
a
4 M
(
1
1
2
2
0 The yellow residue was dissolved in dry DMF (15 mL), DIPEA (0.32 mL, 1.84 mmol) and 4 Å molecular sieves (~0.5 g)
added. After stirring at rt. for 30 mins, 3-hydroxy-5-methoxy-3-(methoxycarbonyl)-5-oxopentanoic acid (0.071 g, 0.324
mmol), HOBt.H O (0.044 g, 0.324 mmol) and EDC.HCl (0.062 g, 0.324 mmol) were added. The resultant mixture was left
2
to stir at rt. for 5 d, filtered, washed with CHCl (10 mL) and the solvent removed in vacuo. The residue was diluted with
3
water (60 mL) and then extracted with DCM (3 × 80 mL). The organic extract was then dried over MgSO , filtered and the
4
5 solvent removed in vacuo. The residue was redissolved in DCM (80 mL) and washed with 0.05 M MeSO H (20 mL), H O
3
2
(
20 mL), 0.05 M NaHCO (20 mL), brine (20 mL) and H O (20 mL). The organic extract was dried over MgSO , filtered
3 2 4
and dried in vacuo to afford a pale yellow solid. The product was triturated in diethyl ether, filtered and washed with
-
1
diethyl ether to afford 160 mg of 6b (75%) as a yellow powder. IR (KBr, cm ) 3426br, 3093w, 3002w, 2951w, 2861w
CH), 1734s (C=O), 1685w, 1623s (C=O), 1587m, 1559w, 1545m, 1493s (C-O), 1478s (C-O), 1437s (C-O), 1387m,
(
1
3
0 1313m, 1261s, 1245s, 1210s, 1167m, 1098m, 1035m. H NMR (CDCl , 400 MHz) δ 8.53 (1H, s), 8.01 (1H, d, J = 13.1
3
H
H-F
4
3
Hz), 7.27 (1H, d, J = 4.9 Hz), 6.53 (1H, t, J = 5.5 Hz), 4.85 (1H, s), 3.89 (3H, s), 3.81 (2H, br s), 3.79 (3H, s), 3.69-
H-F
H-H
3
3
7
.65 (2H, m), 3.66 (3H, s), 3.44-3.42 (1H, m), 3.29-3.20 (4H, m), 3.21-3.20 (2H, m), 2.95-2.60 (4H, m), 2.40 (2H, t, JH-H =
13
.7 Hz), 1.73-1.66 (2H, m), 1.63-1.50 (2H, m), 1.33-1.32 (2H, m), 1.13 (2H, m). C NMR (CDCl , 100 MHz) δ 174.2
3
C
1
(
C=O), 173.1 (C=O), 171.4 (C=O), 170.6 (C=O), 169.5 (C=O), 166.4 (C=O), 153.5 (ipso-Ar, d, J = 247 Hz), 148.6
C-F
2
3
2
5 (CH), 144.2 (ipso-Ar, d, J = 10.7 Hz), 138.1 (ipso-Ar), 123.6 (ipso-Ar, d, J = 6.9 Hz), 113.6 (CH, d, J = 23.0 Hz),
C-F
C-F
C-F
1
10.3 (ipso-Ar), 105.3 (CH), 74.0 (4º-C), 53.2 (CH ), 52.2 (CH ), 52.1 (CH ), 50.6 (CH ), 49.8 (CH ), 45.5 (CH ), 44.3
3 3 3 2 2 2
19
(
CH ), 43.1 (CH ), 41.5 (CH ), 39.1 (CH ), 34.1 (CH), 32.5 (CH ), 29.0 (CH ), 22.1 (CH ), 8.3 (CH x2). F NMR (CDCl ,
2 2 2 2 2 2 2 2 3
+
3
6
76 MHz) δ -123.88 (dd, J = 13.5, 7.6 Hz). m/z (ESI); 647 ([M+H] , 100 %); HRMS (ESI) Calc. for C H N O F
F H-F 31 40 4 10
+
47.2723. Found [M+H] , 647.2723 (0.1 ppm error).

2
-[({5-[4-(3-Carboxy-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)piperazin-1-yl]-5-
oxopentycarbamoyl)methyl]-2-hydroxybutanedioic acid (4)
To a solution of 0.1 M Bu NOH (6.2 mL, 0.620 mmol; made by dilution of 1 M TBAH in MeOH with H O) was added
4
2
5
1,4-dimethyl 2-{[(5-{4-[1-cyclopropyl-6-fluoro-3-(methoxycarbonyl)-4-oxo-1,4-dihydroquinolin-7-yl]piperazin-1-yl}-5-
oxopentyl)carbamoyl]methyl}-2-hydroxybutanedioate 6b (0.10 g, 0.155 mmol) and the mixture stirred at rt. for 18 h.
The pH of the solution was reduced to pH 3.5 through addition of 0.2 M pyruvic acid (~3.1 mL). The resultant precipitate
was filtered, washed with ice-cold H O (5 mL) and CHCl (200 mL) and dried on the vacuum line to afford 47 mg of 4
2
3
-
1
(
50%) as a cream solid. IR (KBr, cm ) 3397br (OH), 3092w (CH), 2931w (CH), 1719s (C=O), 1629s (C=O), 1558w,
1
1
2
0 1539w, 1490s, 1472s, 1384m, 1340m, 1299w, 1250s (C-O), 1215m (C-O), 1184w, 1146w, 1027w.
1
4
H NMR (d -DMSO, 400 MHz) δ 15.19 (1H, br s), 12.40 (2H, br s), 8.66 (1H, s), 7.96-7.90 (2H, m, H), 7.57 (1H, d, J
H-F
6
H
=
5.8 Hz), 3.82 (1H, br s), 3.68 (4H, br s), 3.27 (4H, br s), 3.05 (2H, br s), 2.73-2.50 (4H, m), 2.37 (2H, br s), 1.52 (2H, br
1
3
s), 1.43 (2H, br s), 1.32 (2H, s), 1.18 (2H, s). C NMR (d -DMSO, 100 MHz) δ 176.4 (C=O), 171.3 (C=O), 170.8 (C=O),
6
C
1
1
69.5 (C=O), 166.0 (C=O), 153.0 (ipso-Ar, d, J = 249 Hz), 148.1 (CH), 144.9 (ipso-Ar), 139.2 (ipso-Ar), 118.3 (ipso-
C-F
3
5 Ar), 111.0 (CH, d, J = 22.3 Hz), 106.8 (ipso-Ar), 106.7 (CH), 72.9 (4º-C), 49.7 (CH ), 49.3 (CH ), 44.6 (CH ), 43.1
C-F
2
2
2
19
(
CH ), 43.0 (CH ), 40.6 (CH ), 38.2 (CH ), 35.9 (CH), 31.9 (CH ), 28.7 (CH ), 22.2 (CH ), 7.6 (CH x2). F NMR (d6-
2 2 2 2 2 2 2 2
+
DMSO, 376 MHz) δ -121.85 (dd, J
= 12.6, 6.9 Hz). m/z (ESI); 605 ([M+H] , 100 %); HRMS (ESI) Calc. for
H-F
F
+
C H N O F 605.2253. Found [M+H] , 605.2258 (0.7 ppm error).
2
8
34
4
10
0 Acknowledgements
We thank the University of York for financial support, Leeanne Rayner for technical assistance with the antimicrobial
assays and Accelrys for provision of licenses to software. This work was part-funded by the Wellcome Trust [ref:
0
97829/Z/11/A] and doctoral training grant award (WT095024MA).
2
3
5 References and notes
1
2
3
4
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0 5. T. A. Wencewicz, U. Möllmann, T. E. Long and M. J. Miller, Biometals, 2009, 22, 633-648.

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0. A. Souto, M. A. Montaos, M. Balado, C. R. Osorio, J. Rodrígez, M. L. Lemos and C. Jiménez, Bioorg. Med. Chem., 2013, 21, 295-
3
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1. S. R. Md-Saleh, E. C. Chilvers, K. G. Kerr, S. J. Milner, M. Snelling, J. P. Weber, G. H. Thomas, A.-K. Duhme-Klair and A.
Routledge, Bioorg. Med. Chem. Lett., 2009, 19, 1496-1498.
0 12. S. J. Milner, A. Seve, A. M. Snelling, G. H. Thomas, K. G. Kerr, A. Routledge and A.-K. Duhme-Klair, Org. Biomol. Chem., 2013,
1
1
2
2
3
1
1
1
6
1, 3461-3468.
3. C. Hennard, Q. C. Truong, J.-F. Desnottes, J.-M. Paris, N. J. Moreau and M. A. Abdallah, J. Med. Chem., 2001, 44, 2139-2151.
4. F. Rivault, C. Liébert, A. Burger, F. Hoegy, M. A. Abdallah, I. J. Schalk and G. L. A. Mislin, Bioorg. Med. Chem. Lett., 2007, 17,
40-644.
5 15. S. Noël, V. Gasser, B. Pesset, F. Hoegy, D. Rognan, I. J. Schalk and G. L. A. Mislin, Org. Biomol. Chem., 2011, 9, 8288-8300.
1
1
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