Staphyloferrin A as siderophore-component in fluoroquinolone-based Trojan horse antibiotics

Article abstract of DOI:10.1039/c3ob40162f

A series of fluoroquinolone conjugates was synthesised by linking the carboxylic acid functionality of the carboxylate-type siderophore staphyloferrin A and its derivatives to the piperazinyl nitrogen of ciprofloxacin and norfloxacin via amide bond formation. Four siderophore-drug conjugates were screened against a panel of bacteria associated with infection in humans. Whilst no activity was found against ciprofloxacin- or norfloxacin-resistant bacteria, one of the conjugates retained antibacterial activity against fluoroquinolone-susceptible strains although the structure of its lysine-based siderophore component differs from that of the natural siderophore staphyloferrin A. In contrast, three ornithine-based siderophore conjugates showed significantly reduced activity against strains that are susceptible to their respective parent fluoroquinolones, regardless of the type of fluoroquinolone attached or chirality at the ornithine C_α-atom. The loss of potency observed for the (R)- and (S)-ornithine-based ciprofloxacin conjugates correlates with their reduced inhibitory activity against the target enzyme DNA gyrase. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3ob40162f

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Staphyloferrin A as siderophore-component in
fluoroquinolone-based Trojan horse antibiotics†
Cite this: DOI: 10.1039/c3ob40162f
Stephen J. Milner,a Alexandra Seve,a Anna M. Snelling,b Gavin H. Thomas,c
Kevin G. Kerr,b,d Anne Routledge*a and Anne-Kathrin Duhme-Klair*a
A series of fluoroquinolone conjugates was synthesised by linking the carboxylic acid functionality of the
carboxylate-type siderophore staphyloferrin A and its derivatives to the piperazinyl nitrogen of cipro-
floxacin and norfloxacin via amide bond formation. Four siderophore–drug conjugates were screened
against a panel of bacteria associated with infection in humans. Whilst no activity was found against
ciprofloxacin- or norfloxacin-resistant bacteria, one of the conjugates retained antibacterial activity
against fluoroquinolone-susceptible strains although the structure of its lysine-based siderophore com-
ponent differs from that of the natural siderophore staphyloferrin A. In contrast, three ornithine-based
siderophore conjugates showed significantly reduced activity against strains that are susceptible to their
respective parent fluoroquinolones, regardless of the type of fluoroquinolone attached or chirality at the
ornithine Cα-atom. The loss of potency observed for the (R)- and (S)-ornithine-based ciprofloxacin conju-
gates correlates with their reduced inhibitory activity against the target enzyme DNA gyrase.
Received 24th January 2013,
Accepted 27th March 2013
DOI: 10.1039/c3ob40162f
www.rsc.org/obc
To date, antibiotic conjugates designed to overcome mem-
brane permeation-based drug access problems have mainly
Introduction
The development of Trojan horse antibiotics that consist of an utilised siderophores of the catecholate- and hydroxamate-type
antimicrobial agent linked to a bacterial iron carrier (sidero- as delivery vehicles.1,7 However, using carboxylate-type sidero-
phore) has attracted much attention due to the potential of phores, such as staphyloferrin A (Fig. 1), can be advantageous
these conjugates to promote active drug uptake and to evade for certain applications, as this type of siderophore remains an
membrane-associated resistance mechanisms.1–4
efficient iron chelator in mildly acidic environments, due to
We have chosen fluoroquinolones, such as ciprofloxacin the low pKa values of the carboxylic acid groups, whilst
and norfloxacin, as antimicrobial components since they are
widely used for the treatment of a broad range of infections.
However, with their increased use the incidence of resistance
in almost all bacteria that fluoroquinolones are used against
has emerged in both medical and veterinary settings.5 The
main resistance mechanisms are alterations of the drug target
enzymes, DNA gyrase and topoisomerase IV, and alterations
that limit permeation of the drug to the target. The latter result
from mutations that either increase the expression of efflux
pumps or down-regulate the expression of outer membrane
porins that are involved in fluoroquinolone uptake, or both.6
aDepartment of Chemistry, University of York, Heslington, York YO10 5DD, UK.
E-mail: anne.duhme-klair@york.ac.uk; Fax: +44 (0)1904 32 2516;
Tel: +44 (0)1904 32 2587; E-mail: anne.routledge@york.ac.uk; Fax: (+1904) 32 2516;
Tel: (+1904) 32 4540
bBradford Infection Group, University of Bradford, Bradford, BD7 1DP, UK
Fig. 1 Top: structure of the (S),(R),(R)-form of staphyloferrin A (left) and its Fe(III)-
cDepartment of Biology, University of York, Heslington, York YO10 5YW, UK
complex shown in stick representation (right). Bottom: binding of the Fe(III)-
dDepartment of Microbiology, Harrogate District Hospital, Lancaster Park Road,
complex to the open (left) and closed (right) form of its receptor HtsA shown in
spacefill representation (3LHS·pdb and 3LI2·pdb, respectively, figures generated
with PyMOL, Fe orange, O red, N blue, C grey, H omitted for clarity, H-bonds
dashed).
Harrogate, HG2 7SX, UK
†Electronic supplementary information (ESI)
10.1039/c3ob40162f
available. See DOI:
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catecholate and hydroxamate siderophores show significantly
reduced iron(III)-affinity in acidic environments.
We have thus designed a series of novel staphyloferrin-
based Trojan horse conjugates. These have the potential to
target staphylococci, such as Staphylococcus aureus, since these
are known to secrete staphyloferrin-type siderophores. S.
aureus can colonise human skin, the nares, and the gut and
some of the most problematic healthcare-associated infec-
tions, including surgical site infections, bacteraemias and
lower respiratory tract infections, are caused by methicillin (or
colloquially multidrug)-resistant S. aureus (MRSA).
Scheme 1 Acid-catalysed cyclisation of staphyloferrin A.
Advantages of staphyloferrin A are its hexadentate nature,
which confers high affinity for iron(III) and its hydrophilicity,
which improves the water solubility of its conjugates. Carboxy-
late siderophores, such as the staphyloferrins, are most com-
petitive under slightly acidic conditions. Accordingly, the iron-
transport optimum of rhizoferrin, a di-citrate siderophore
structurally related to staphyloferrin A, lies close to the average
pH of 5.5 of the skin.8 Trojan horse antibiotics based on sta-
phyloferrin A could therefore have advantages in topical appli-
cations, such as the treatment of skin infections.
screened as such. In addition, a conjugate containing an
(S)-lysine-backbone was prepared and tested for comparison.
In this way, the effect of the backbone length and chirality on
the antibacterial activities could be investigated.
Briefly, after Boc-protection of the two amino groups of
ornithine or lysine,14 the protected diamino acids 2a–c were
reacted with the methyl esters of ciprofloxacin (3) and norflox-
acin (4) using and EDC-mediated coupling procedure to give
5a–d in moderate to good yields.15 Subsequent Boc-deprotec-
tion16 provided the hydrochloride salts 6a–d which were
reacted in situ with two equivalents of 1,3-dimethyl citrate
(7)17,18 to produce the methyl-protected pre-cursors 8a–d. The
isolation and characterisation of 8a–d by ESI mass spectro-
metry and NMR spectroscopy, however, revealed the presence
of cyclised by-products analogous to those shown in Scheme 1.
Since repeated purification attempts by column chromato-
graphy led to the production of more cyclised by-products, it
was decided to follow the amide coupling step immediately
with based-catalysed ester hydrolysis in situ in order to re-open
any cyclised succinimide rings. A careful optimisation of the
conditions used in the final neutralisation step was required,
in order to avoid both re-cyclisation and cleavage of the citrate-
based amide bonds. The latter were found to be susceptible to
hydrolysis by strong mineral acids. Neutralisation with 0.2 M
methanesulfonic acid furnished conjugates 9a–d in low to
moderate yields without formation of hydrolysis or cyclisation
products.
Results and discussion
Design and synthesis of conjugates
There is currently no published chemical synthesis of staphylo-
ferrin A. Native staphyloferrin A has three chiral centres, the
Cα-atom of the ornithine backbone, which has (R)-configur-
ation and the quaternary C-atoms of the two citryl-moieties.
Jung et al. proposed that the two citryl-moieties have (S),(R)-
configuration,9 whilst Drechsel and Winkelmann suggested
(S),(S)-configuration based on circular dichroism spectra.10 In
a recent crystallographic study on ferric staphyloferrin A
bound to the open and closed form of its receptor protein
HtsA, the observed electron density is most consistent with the
solvent-exposed citryl moiety having (R)- and the buried citryl-
moiety having (S)-configuration (Fig. 1).11 The crystal structure
also revealed that the carboxylate group of the ornithine back-
bone is solvent exposed and therefore a suitable site for
conjugation.
Antibacterial activity
In addition to the stereochemical challenges, the synthesis
of staphyloferrin A is complicated by the fact that the citrate To assess the influence of the siderophore moiety on the anti-
moieties can undergo cyclisation under acidic conditions to microbial activity of the parent fluoroquinolones, the conju-
form succinimides (Scheme 1). Cyclisation has been observed gates were screened against a collection of 19 reference and
by 13C NMR spectroscopy in the naturally occurring sidero- clinical isolates of bacteria associated with infection in
phores staphyloferrin A9 and rhizoferrin.12
humans (Tables 1 and 2). In addition, two representative
To access a series of fluoroquinolone conjugates of staphylo- examples of compound series 6, one with the methyl-ester of
ferrin A and related derivatives, we used the synthetic strategy ciprofloxacin as the antibiotic component (6c) and one with
shown in Scheme 2, thereby capitalising on our previous the methyl-ester of norfloxacin as the antibiotic component
work on citrate–ciprofloxacin conjugates.13 Commercially avail- (6d) were also screened, using the methyl-esters of ciproflox-
able racemic dimethylcitrate was used in combination with acin (3) and norfloxacin (4) as controls. As summarised in
both (R)- and (S)-ornithine. Consequently, two mixtures of four Table S1 (ESI†), the methyl-esters 3 and 4 are inactive. The lack
diastereoisomers containing an enantiomerically pure (R)- or of antimicrobial activity is consistent with the fluoroquinolone
(S)-ornithine backbone in combination with (S),(S)-, (R),(R)-, pharmacophore relying on the free carboxylic acid group,
(S),(R′)- or (R),(S′)-configured citryl moieties were obtained and which is required for target binding. Koga et al. also reported
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Scheme 2 Synthesis of staphyloferrin–fluoroquinolone conjugates. Reagents and conditions: (i) (Boc2)O, NaOH, H2O/1,4-dioxane, 50 °C, 3 h, 77–98%; (ii) EDC·HCl,
HOBt·H2O, DIPEA, DMF, r.t., 7 d, 60–80%; (iii) 4 M HCl in MeOH, 0 °C–r.t., 1 h; (iv) EDC·HCl, HOBt·H2O, DIPEA, DMF, r.t., 24–72 h; (v) NaOH, r.t., 24 h, 13–22%.
Table 1 Activity of ciprofloxacin conjugates 9a–c in a disc diffusion assay
Table 2 Activity of the norfloxacin conjugate 9d in a disc diffusion assay
Diameter of zone of
inhibition (mm)
Diameter of zone of
inhibition (mm)
9aa
9ba
1
9ca
1
9da
Ciproa
Control 1
Norfloxacina
Control
Bacterial strain
2
2
2
Bacterial strain
1
2
Staphylococcus aureus (Oxford) NCTC 6571 29
R
R
R
R
R
9
R
12 R
R
R
R
R
R
R
R
R
R
R
Staphylococcus aureus (Oxford) NCTC 6571
22
26
R
24
R
24
23
26
30
35
R
R
R
R
R
R
R
R
R
R
14
R
R
R
R
R
R
R
13
R
R
R
R
R
R
R
R
R
R
17
R
R
R
R
R
R
R
15
R
Staphylococcus aureus NCTC 10399
30
R
28
R
27
30
30
13 13 Staphylococcus aureus NCTC 10399
R R
Staphylococcus aureus HG-1b,c
R
Staphylococcus aureus HG-1b,c
Staphylococcus aureus NCTC 13142b
Staphylococcus aureus NCTC 13143b,c
Staphylococcus aureus BIG 0052b,c
Staphylococcus epidermidis NCTC 11047
Staphylococcus epidermidis NCTC 2749
13 R 12 11 Staphylococcus aureus NCTC 13142b
R
R
R
R
R
R
8
R
R
Staphylococcus aureus NCTC 13143b,c
12 12 Staphylococcus aureus BIG 0052b,c
11 R
R
22 25 R
11 11 Staphylococcus epidermidis NCTC 11047
R
10 12 12 Staphylococcus epidermidis NCTC 2749
17 15 Staphylococcus haemolyticus NCTC 11042
Staphylococcus haemolyticus NCTC 11042 33
R
Escherichia coli NCTC 10418
40
R
R
20 19 18 19 25 28 Escherichia coli NCTC 10418
R
R
Escherichia coli BIG 0046c
R
R
R
R
R
R
R
R
R
R
R
Escherichia coli BIG 0046c
Escherichia coli BIG 0051c
Escherichia coli BIG 0051c
R
Pseudomonas aeruginosa NCTC 6749
Pseudomonas aeruginosa BIG 0039
Pseudomonas aeruginosa BIG 0037
Pseudomonas aeruginosa NCTC 10662
Pseudomonas aeruginosa BIG 0063
Serratia marcescens NCTC 1377
Burkholderia cepacia NCTC 10744
30
30
31
31
30
29
25
12 19 R
12 12 Pseudomonas aeruginosa NCTC 6749
28
28
29
26
25
27
22
R
R
R
R
R
R
11 R
10 10 11 12 Pseudomonas aeruginosa BIG 0039
R
R
R
R
R
R
Pseudomonas aeruginosa BIG 0037
11 12 Pseudomonas aeruginosa NCTC 10662
11 11 Pseudomonas aeruginosa BIG 0063
R
R
20 21 16 15 21 23 Serratia marcescens NCTC 1377
Burkholderia cepacia NCTC 10744
R
R R R 9 9
R = resistant, zone of inhibition = 0 mm.a 5 μg ciprofloxacin (cipro) R = resistant, zone of inhibition = 0 mm.a 5 μg norfloxacin discs and
discs and equimolar conjugate discs were used in the assay. equimolar conjugate discs were used in the assay. b Methicillin-
b Methicillin-resistant S. aureus (MRSA). c Strains of clinical origin resistant S. aureus (MRSA). c Strains of clinical origin defined as
defined as resistant to ciprofloxacin by CLSI criteria.20
resistant to ciprofloxacin by CLSI criteria.20
that the methyl-ester of norfloxacin is inactive.19 Compounds
For the siderophore conjugates of series 9, no activity was
6c and 6d show no activity against the majority of strains observed against ciprofloxacin- or norfloxacin-resistant strains.
tested, with the exception of Escherichia coli NCTC 10418 in The ornithine-based conjugates 9a, 9b and 9d show drastically
case of 6c and Escherichia coli NCTC 10418, Staphylococcus hae- reduced activity against strains that were susceptible to their
molyticus NCTC 11042 and Staphylococcus aureus NCTC 10399 respective parent fluoroquinolone, as indicated by smaller or
in case of 6d. The lack of activity can be explained by the fact no zones of inhibition in a disc diffusion assay. The loss of
that the conjugates contain the antibiotic component in its activity occurs for both the ciprofloxacin and norfloxacin con-
methyl-protected and hence inactivated form.
jugates and regardless of the chirality at the Cα-atom of the
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ornithine backbone, which has (R)-configuration in the natural still active, their activity against DNA gyrase is reduced, poten-
siderophore staphyloferrin A. To investigate if this significant tially due to steric clashes of the modified drug when binding
loss of activity is due to an inability to inhibit the target to its binding site in the enzyme or electrostatic repulsion. The
enzyme, DNA gyrase inhibition assays were performed with 9a effect is independent of the chirality at the ornithine Cα-atom.
and 9b (vide infra).
These observations suggest that the introduction of a spacer
On the other hand, the (S)-lysine-based ciprofloxacin conju- unit between the fluoroquinolone and siderophore unit and/or
gate 9c retains some activity against 12 of the 14 ciprofloxacin the use of a bio-labile linker may give rise to improved DNA
susceptible strains (including 5 of the 6 susceptible staphylo- gyrase inhibitory activity. Bio-labile linkers have recently
cocci), albeit with reduced zone diameters (Table 1). This is shown promise in conjugates involving fluoroquinolones and
surprising since the structure of the siderophore component both the hydroxamate siderophore desferrioxamine B21 and
in 9c differs significantly from that of natural staphyloferrin the pseudomonal siderophore pyochelin.22
A. Firstly, the siderophore backbone consists of lysine instead
of ornithine and the spacer thus contains an additional
methylene group and secondly the Cα-atom of the backbone
has (S)-rather than (R)-configuration. Although 9c (S-lysCpf)
Summary and conclusions
has a significantly higher molecular mass (918 g mol−1) than A series of four structurally related fluoroquinolone conjugates
its parent drug ciprofloxacin (331 g mol−1), this result suggests has been synthesised by linking the carboxylic acid functional-
that 9c is still able to reach and inhibit the target enzyme ity of staphyloferrin A and its derivatives to the piperazinyl
within the cytoplasm.
nitrogen of the parent drug via amide bond formation. The
However, the significant drop in fluoroquinolone potency resulting siderophore–fluoroquinolone conjugates 9a–d were
upon conjugation of staphyloferrin A and its derivatives did screened against a panel of bacteria associated with infection
not merit a further evaluation of the enantiomerically pure in humans. No activity was observed against ciprofloxacin- or
components containing (S),(S)-, (R),(R)-, (S),(R′)- or (R),(S′)-con- norfloxacin-resistant bacteria. Surprisingly, 9c retained some
figured citryl moieties. Hence no attempt was made to separate antibacterial activity although the structure of its (S)-lysine-
diastereomeric mixtures 9a–d.
based siderophore component differs significantly from that
of natural siderophore staphyloferrin A, which contains an
(R)-ornithine backbone. In contrast, the three ornithine-based
conjugates 9a,b,d showed significantly reduced activity against
strains that are susceptible to their respective parent fluoro-
quinolones, regardless of the type of fluoroquinolone attached
or chirality at the ornithine Cα-atom. The (R)- and (S)-ornithine-
based ciprofloxacin conjugates 9a and 9b, respectively, were
found to retain some inhibitory activity against the target
enzyme DNA gyrase, although activity was significantly
reduced. Future studies will be directed towards the enhance-
ment of the DNA inhibitory activity of related conjugates
through the introduction of a spacer between the fluoroquino-
lone and the siderophore and the use of bio-labile linkers
designed to release the drug from the siderophore once trans-
ported into the cell.
Gyrase assay
To assess whether the modification of ciprofloxacin with (R)-
and (S)-ornithine-based staphyloferrin derivatives alters the
ability of the antimicrobial to inhibit the activity of DNA
gyrase, we measured DNA gyrase inhibitory activity of 9a and
9b using a commercial DNA gyrase supercoiling assay (Inspira-
lis). In this assay a non-supercoiled template plasmid is super-
coiled through the action of the DNA gyrase which results in a
detectable mobility shift of the plasmid DNA when electro-
phoresed on an agarose gel. Using a concentration of cipro-
floxacin that gives complete inhibition of DNA gyrase activity
(10 μM) (Fig. 2), we could also see significant inhibition of
gyrase activity by both 9a (R-ornCpf) and 9b (S-ornCpf),
although they did not achieve complete inhibition at this con-
centration. The data suggests that whilst the compounds are
Experimental section
Materials and methods
Chemical reagents were purchased from Aldrich or Fluka and
were used as supplied. 1,3-Dimethyl citrate (7) and the methyl
esters of ciprofloxacin (3) and norfloxacin (4) were prepared as
described previously.13 Solvents were supplied by Aldrich or
Fisher Scientific and used as received or dried over 3 or 4 Å
molecular sieves prior to use. Dialysis tubing (Spectra/Por® CE
Float-A-Lyzer® G2, approximate molecular weight cut-off
100–500 Daltons) was obtained from Fisher Scientific. 1H NMR
(399.785 MHz), 13C NMR (100.535 MHz) and 19F NMR
(376.152 MHz) spectra were acquired on a Jeol ECX400 or
ECS400 spectrometer in the stated deuterated solvents.
Fig. 2 Ethidium bromide stained agarose gel showing data from DNA gyrase
assays. Lane 1 is the template (originally in the open complex [OC] form) which
has been acted upon by DNA gyrase forming the supercoiled form [SC] and lane
2 is template alone. Lanes 3–5 contain template, gyrase and 10 μM of either
ciprofloxacin (lane 3), 9b (S-ornCpf, lane 4) or 9a (R-ornCpf, lane 5).
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Chemical shifts (δ) are reported in parts per million (ppm) EDC·HCl (0.88 mmol) were added. The resultant mixture was
1
using the solvent residual peak as an internal standard for H left to stir at room temperature for 5 days. The precipitated
and 13C NMR spectra: 7.26 ppm and 77.16 ppm respectively urea by-product was filtered off and washed with CHCl3
for CDCl3; 2.50 ppm and 39.52 ppm respectively for (D3C)2SO; (20 mL). The filtrate was concentrated in vacuo to a volume of
3.31 ppm and 49.00 ppm respectively for CD3OD. Spectra per- approximately 1 mL, diluted with water (60 mL) and then
formed in D2O were spiked with CD3CN and its solvent extracted with DCM (3 × 30 mL). The organic extracts were
residual peaks used as an internal standard for 13C NMR: 1.32/ combined, washed with water (50 mL), aqueous methanesulfo-
118.26 ppm. Fluorobenzene was used as an internal standard nic acid (20 mL, 0.05 M), aqueous sodium hydrogen carbonate
for 19F NMR spectra, taken as −113.150 ppm with reference to (20 mL, 0.05 M) and water (20 mL). The organic extract was
CFCl3 as 0 ppm. Coupling constants (J) are reported in Hertz dried over MgSO4, filtered and dried in vacuo to afford com-
(Hz). All 13C experiments were confirmed using DEPT experi- pounds 8 as brown glass.
ments. Electrospray ionisation (ESI) mass spectra were
The appropriate compound 8 was dissolved in aqueous 0.25
acquired on a Thermo-Finnigan LCQ mass spectrometer. Infra- M NaOH (3 mL, 0.75 mmol) and left to stir at room temp-
red spectra were recorded on a Thermo Nicolet Avatar FTIR erature for 24 hours. The pH of the mixture was adjusted to 7
spectrometer as KBr discs or as a solution in the stated anhy- through the addition of 0.2 M methanesulfonic acid (∼1.5 mL)
drous solvent. Melting points were acquired on a Stuart Scien- and the mixture was dialysed against water at room temp-
tific SMP3 apparatus and are uncorrected.
General procedure for the synthesis of 2a–c. To a stirred The sample was dried in vacuo to afford 9 as bronze powder.
solution of the respective diaminocarboxylic acid (7.00 mmol)
erature for 48 hours, changing the water at 2, 8 and 24 hours.
Data for (2R)-2,5-bis({[(tert-butoxy)carbonyl]amino})pentanoic
and sodium hydroxide (14.10 mmol) in deionised water acid (2a). Molecular formula: C15H28N2O6, molecular weight:
(13 mL) was added drop-wise a solution of di-tert-butyl dicar- 332.397 g mol−1. Yield = 1.80 g, 5.42 mmol, 77%; Rf 0.55
bonate (17.5 mmol) in dry 1,4-dioxane (25 mL) and the result- (MeCN). IR (0.03 M DCM, cm−1) 3438br (OH), 3057w, 3002w
ing mixture was left to stir at 50 °C for 3 hours. The mixture (CH), 2978m (CH), 2931m (CH), 2866w (CH), 1708s (CvO),
was concentrated to ∼6 mL in vacuo, diluted with H2O (20 mL) 1501s (C–O), 1453m, 1446m, 1409m, 1392m, 1364s (C–O),
1
and washed with cyclohexane (3 × 25 mL). The pH of the 1279m, 1245m, 1160s, 1099w, 1048w, 1014w. H NMR (CDCl3,
3
aqueous layer was reduced to ∼3 through addition of aqueous 400 MHz) δH 9.21 (1H, br s), 5.25 (1H, d, JH–H = 7.40 Hz), 4.80
hydrochloric acid (∼8 mL, 1 M). The resultant precipitate was (1H, br s), 4.32 (1H, br s), 3.14–3.13 (2H, br m), 1.87–1.87 (1H,
extracted with ethyl acetate (3 × 25 mL), the organic layer dried br m), 1.73–1.61 (1H, br m), 1.59–1.56 (2H, m), 1.43 (18H). 13C
over MgSO4 filtered and the solvent removed in vacuo to afford NMR (CDCl3, 100 MHz) δC 175.93 (CvO), 156.57 (CvO),
2a–c as colourless oil or gel.
155.83 (CvO), 80.25 (4°-C), 79.74 (4°-C), 53.09 (CH), 40.07
General procedure for the synthesis of 5a–d. To a stirred (CH2), 29.96 (CH2), 28.52 (CH3), 28.46 (CH3), 26.17 (CH2). m/z
solution of the methyl ester of ciprofloxacin (3) or norfloxacin (ESI); 333 ([M + H]+, 100%); HRMS (ESI) Calc. for C15H29N2O6
(4) (1.45 mmol), the appropriate compound 2 (1.45 mmol) and 333.2020. Found [M + H]+, 333.2025 (1.8 ppm error).
HOBt·H2O (0.20 g, 1.45 mmol) in dry DMF (30 mL) were added
Data for (2S)-2,5-bis({[(tert-butoxy)carbonyl]amino})pentanoic
DIPEA (260 μL, 1.49 mmol) and EDC·HCl (0.28 g, 1.45 mmol). acid (2b). Molecular formula: C15H28N2O6, molecular weight:
The resultant mixture was left to stir at room temperature for 332.397 g mol−1. Yield = 2.27 g, 6.83 mmol, 98%; Rf 0.42
7 days and the solvent removed in vacuo. The residue was (MeCN). IR (0.03 M DCM, cm−1) 3443br (OH), 3067w (CH),
dissolved in water (60 mL) and then extracted with DCM 3003w (CH), 2981w (CH), 2932w (CH), 1712s (CvO), 1504m,
(3 × 80 mL). The organic extract was then washed with water 1454w, 1393w, 1368m, 1249m (C–O), 1165s, 1100w, 1053w,
(50 mL), aqueous hydrochloric acid (50 mL, 0.05 M), aqueous 1019w. 1H NMR (CDCl3, 400 MHz) δH 7.57 (1H, br s), 5.27 (1H,
3
sodium hydrogen carbonate (50 mL, 0.05 M), brine (50 mL) d, JH–H = 6.72 Hz), 4.81 (1H, br s), 4.32 (1H, br s), 3.14–3.11
and water (50 mL). The organic extract was dried over MgSO4, (2H, br m), 1.87 (1H, br s), 1.72–1.63 (1H, m), 1.61–1.51 (2H,
filtered and the volume of the filtrate was reduced in vacuo to m), 1.43 (18H, s). 13C NMR (CDCl3, 100 MHz) δC 175.87
afford a yellow residue. The residue was dissolved in DCM and (CvO), 156.44 (CvO), 155.80 (CvO), 80.11 (4°-C), 79.57
precipitated with diethyl ether, filtered and washed with (4°-C), 53.09 (CH), 40.06 (CH2), 29.88 (CH2), 28.46 (CH3), 28.40
diethyl ether to afford a yellow powder.
(CH3), 25.98 (CH2). m/z (ESI); 333 ([M + H]+, 100%); HRMS
General procedure for the synthesis of 9a–d. To a stirred (ESI) Calc. for C15H29N2O6 333.2020. Found [M + H]+, 333.2026
solution of ice-cold 4 M HCl in MeOH (30 mL) was added the (1.9 ppm error).
respective compound 5 (0.44 mmol) and the mixture left to stir
Data for (2S)-2,6-bis({[(tert-butoxy)carbonyl]amino})hexanoic
at room temperature for 1 hour. The solvent and HCl was acid (2c). Molecular formula: C16H30N2O6, molecular weight:
removed in vacuo to afford the respective compound 6 as a 346.424 g mol−1. Yield = 2.01g, 5.80 mmol, 83%. IR (0.03 M
yellow residue.
DCM, cm−1) 3446br (OH), 3325br (NH), 1711s (CvO), 1681m
The appropriate compound 6 was dissolved in dry DMF (CvO). 1H NMR (CDCl3, 400 MHz) δH 5.27 (1H, br s), 4.68 (1H,
(20 mL) and 4 Å molecular sieves (∼0.5 g) added. After stirring br s), 4.29 (1H, br s), 3.12–3.11 (2H, br m), 1.84 (1H, br s),
at room temperature for 30 minutes, 1,3-dimethyl citrate (7) 1.73–1.71 (1H, br m), 1.50 (2H, br s), 1.44 (20H, s). 13C NMR
(0.88 mmol), HOBt·H2O (0.88 mmol), DIPEA (4.00 mmol) and (CDCl3, 100 MHz) δC 176.37 (CvO), 156.44 (CvO), 155.96
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4
(CvO), 80.14 (4°-C), 79.49 (4°-C), 53.34 (CH), 40.21 (CH2), (1H, s), 7.96 (1H, d, JH–F = 13.1 Hz), 7.24 (1H, d, JH–F
=
3
32.13 (CH2), 29.62 (CH2), 28.52 (CH3), 28.46 (CH3), 22.51 7.04 Hz), 5.44 (1H, d, JH–H = 8.52 Hz), 4.68 (1H, br s),
(CH2). m/z HRMS (ESI) Calc. for C16H31N2O6 347.2177. Found 4.63–4.58 (1H, m), 3.95–3.92 (1H, br m), 3.87 (3H, s), 3.80–3.77
[M + H]+, 347.2186 (2.7 ppm error).
(1H, br m), 3.70–3.69 (2H, br m), 3.44–3.39 (1H, m), 3.26–3.18
Data for 5a. Molecular formula: C33H46N5O8F, molecular (4H, br m), 3.16–3.03 (2H, br m), 1.72–1.66 (1H, m), 1.59–1.54
weight: 659.756 g mol−1. Yield = 0.76 g, 1.15 mmol, 79%; Rf (1H, m), 1.54–1.46 (2H, m), 1.42 (11H, s), 1.39 (9H, s),
0.58 (6 : 1 DCM : MeOH); m.p. 165.5–167.9 °C. IR (KBr, cm−1
)
1.32–1.29 (2H, m), 1.14–1.10 (2H, m). 13C NMR (CDCl3,
3430br (NH), 3340br (NH), 2977m (CH), 2932m (CH), 2867w 100 MHz) δC 173.03 (CvO), 170.91 (CvO), 166.29 (CvO),
1
(CH), 1701s (CvO), 1648s (CvO), 1624s (CvO), 1589m, 1495s, 156.17 (CvO), 155.74 (CvO), 153.38 (ipso-Ar, d, JC–F
=
2
1478s, 1440s, 1391m, 1366m, 1349m, 1329m, 1316m, 1247s 249 Hz), 148.55 (CH), 143.99 (ipso-Ar, d, JC–F = 10.7 Hz),
3
(C–O), 1211m (C–O), 1165s (C–O), 1103m, 1088m, 1026m. 1H 138.01 (ipso-Ar), 123.59 (ipso-Ar, d, JC–F = 6.91 Hz), 113.47
3
2
3
NMR (CDCl3, 400 MHz) δH 8.55 (1H, s), 8.06 (1H, d, JH–F
=
=
(CH, d, JC–F = 22.2 Hz), 110.13 (ipso-Ar), 105.34 (CH, d, JC–F
=
4
3
13.1 Hz), 7.27 (1H, d, JH–F = 6.12 Hz), 5.41 (1H, d, JH–H
2.30 Hz), 79.88 (4°-C), 79.17 (4°-C), 52.13 (CH3), 50.33 (CH2),
8.56 Hz), 4.67–4.64 (1H, m), 4.62 (1H, br s), 3.98–3.95 (1H, m), 49.87 (CH2), 49.82 (CH), 45.48 (CH2), 41.94 (CH2), 34.66 (CH),
3.91 (3H, s), 3.84–3.81 (1H, m), 3.75–3.66 (2H, m), 3.45–3.40 33.21 (CH2), 29.64 (CH2), 28.49 (CH3), 28.44 (CH3), 22.43
(1H, m), 3.27–3.11 (6H, m), 1.73–1.71 (2H, br m), 1.57–1.57 (CH2), 8.23 (CH2). m/z (ESI); 674 ([M + H]+, 100%); HRMS (ESI)
(2H, br m), 1.43 (9H, s), 1.41 (9H, s), 1.36–1.32 (2H, m), Calc. for C34H49N5O8F 674.3560. Found [M + H]+, 674.3583
1.16–1.12 (2H, m). 13C NMR (CDCl3, 100 MHz) δC 173.07 (2.9 ppm error).
(CvO), 170.80 (CvO), 166.33 (CvO), 156.15 (CvO), 155.66
Data for 5d. Molecular formula: C32H46N5O8F, molecular
1
(CvO), 153.39 (ipso-Ar, d, JC–F = 249 Hz), 148.56 (CH), 143.99 weight: 647.745 g mol−1. Yield = 0.83 g, 1.28 mmol, 88%; Rf
2
(ipso-Ar, d, JC–F = 10.7 Hz), 138.03 (ipso-Ar), 123.63 (ipso-Ar, d, 0.45 (6 : 1 DCM : MeOH); m.p. 83.6–85.6 °C. IR (KBr, cm−1
)
2
3JC–F = 6.90 Hz), 113.53 (CH, d, JC–F = 23.0 Hz), 110.18 3341br (NH), 2976m (CH), 2933m (CH), 2871w (CH), 1701s
(ipso-Ar), 105.34 (CH), 79.94 (4°-C), 79.34 (4°-C), 52.16 (CH3), (CvO), 1622s (CvO), 1560m, 1506m, 1490s, 1448m, 1391m,
50.36 (CH2), 49.86 (CH2), 49.64 (CH), 45.51 (CH2), 41.99 (CH2), 1366m, 1249s (C–O), 1229m (C–O), 1169s, 1103w, 1088w,
1
34.67 (CH), 30.78 (CH2), 28.49 (CH3), 28.43 (CH3), 25.94 (CH2), 1053w, 1025w. H NMR (CDCl3, 400 MHz) δH 8.43 (1H, s), 8.08
8.25 (CH2). m/z (ESI); 660 ([M + H]+, 100%); HRMS (ESI) Calc. (1H, d, JH–F = 12.9 Hz), 6.73 (1H, d, JH–F = 6.64 Hz), 5.42 (1H,
3
4
for C33H47N5O8 660.3403. Found [M + H]+, 660.3423 (2.5 ppm d, JH–H = 8.56 Hz), 4.66 (2H, br s), 4.20 (2H, q, JH–H = 7.24
3
3
error). Hz), 3.95 (1H, br s), 3.90 (3H, s), 3.83–3.79 (1H, br m),
Data for 5b. Molecular formula: C33H46N5O8F, molecular 3.74–3.66 (2H, m), 3.24–3.13 (6H, m), 1.91 (1H, br s), 1.73–1.71
3
weight: 659.756 g mol−1. Yield = 0.57 g, 0.86 mmol, 59%; Rf (1H, br m), 1.58–1.56 (2H, m), 1.53 (3H, t, JH–H = 7.24 Hz),
0.56 (6 : 1 DCM : MeOH); m.p. 166.4–168.1 °C. IR (KBr, cm−1
)
1.42 (9H, s), 1.40 (9H, s). 13C NMR (CDCl3, 100 MHz) δC 173.08
3338br (NH), 2977w (CH), 2931w (CH), 2868w (CH), 1701s (CvO), 170.81 (CvO), 166.64 (CvO), 156.15 (CvO), 155.67
1
(CvO), 1623s (CvO), 1589w, 1495s, 1478s, 1440s, 1400m, (CvO), 153.28 (ipso-Ar, d, JC–F = 249 Hz), 148.50 (CH), 144.28
2
1366m, 1349m, 1329m, 1315m, 1248s (C–O), 1211m (C–O), (ipso-Ar, d, JC–F = 10.7 Hz), 136.15 (ipso-Ar), 124.60 (ipso-Ar, d,
2
1166s (C–O), 1103w, 1088w, 1025m. 1H NMR (CDCl3, 400 MHz) 3JC–F = 6.90 Hz), 114.08 (CH, d, JC–F = 23.0 Hz), 110.41
3
δH 8.52 (1H, s), 8.01 (1H, d, JH–F = 13.1 Hz), 7.25 (1H, d, (ipso-Ar), 104.40 (CH), 79.95 (4°-C), 79.35 (4°-C), 52.24 (CH3),
3
4JH–F = 7.24 Hz), 5.42 (1H, d, JH–H = 8.60 Hz), 4.65 (2H, br s), 50.43 (CH2), 50.02 (CH2), 49.63 (CH), 49.12 (CH2), 45.52 (CH2),
3.97–3.93 (1H, m), 3.89 (3H, s), 3.83–3.80 (1H, br m), 3.74–3.66 42.02 (CH2), 30.82 (CH2), 28.51 (CH3), 28.46 (CH3), 25.98
(2H, br m), 3.45–3.40 (1H, m), 3.26–3.10 (6H, br m), 1.88 (1H, (CH2), 14.57 (CH3). m/z (ESI); 648 ([M + H]+, 100%); HRMS
br s), 1.74–1.71 (1H, br m), 1.58–1.56 (2H, br m), 1.42 (9H, s), (ESI) Calc. for C32H47N5O8F 648.3403. Found [M + H]+,
1.40 (9H, s), 1.35–1.31 (2H, m), 1.15–1.11 (2H, m). 13C NMR 648.3417 (1.7 ppm error).
(CDCl3, 100 MHz) δC 173.07 (CvO), 170.80 (CvO), 166.44
Data for 6a. Molecular formula: C23H30N5O4F·2HCl, mole-
(CvO), 156.14 (CvO), 155.66 (CvO), 153.42 (ipso-Ar, d, 1JC–F
=
cular weight: 532.444 g mol−1. Yield = 0.203 g, 0.38 mmol,
2
249 Hz), 148.59 (CH), 144.01 (ipso-Ar, d, JC–F = 9.97 Hz), 86%. 1H NMR (d6-DMSO, 400 MHz) δH 8.45 (4H, br s), 8.26
3
3
4
138.06 (ipso-Ar), 123.74 (ipso-Ar, d, JC–F = 6.91 Hz), 113.63 (3H, br s) 7.77 (1H, d, JH–F = 13.2 Hz), 7.50 (1H, d, JH–F
=
2
(CH, d, JC–F = 22.2 Hz), 110.29 (ipso-Ar), 105.31 (CH), 79.95 7.3 Hz), 4.51–4.49 (1H, m), 3.76–3.70 (4H, m), 3.73 (3H, s),
(4°-C), 79.35 (4°-C), 52.21 (CH3), 50.37 (CH2), 49.90 (CH2), 3.70–3.66 (1H, m), 3.40–3.22 (4H, m), 2.80–2.79 (2H, m),
49.66 (CH), 45.54 (CH2), 42.01 (CH2), 40.14 (CH2), 34.66 (CH), 1.84–1.80 (2H, m), 1.78–1.67 (2H, m), 1.78–1.67 (2H, m),
30.84 (CH2), 28.50 (CH3), 28.46 (CH3), 25.98 (CH2), 8.29 (CH2). 1.28–1.22 (2H, m), 1.11–1.10 (2H, m). HRMS (ESI) Calc. for
m/z (ESI); 660 ([M + H]+, 100%); HRMS (ESI) Calc. for C23H31N5O4F 460.2355. Found [M + H]+, 460.2371 (3.4 ppm
C33H47N5O8F 660.3403. Found [M + H]+, 660.3410 (0.6 ppm error).
error).
Data for 6b. Molecular formula: C23H30N5O4F·2HCl, mole-
Data for 5c. Molecular formula: C34H48N5O8F, molecular cular weight: 532.444 g mol−1. Yield = 0.075 g, 0.14 mmol,
weight: 673.783 g mol−1. Yield = 0.78 g, 1.16 mmol, 80%; m. 32% 1H NMR (d6-DMSO, 400 MHz) δH 8.47 (3H, br s), 8.44
p. 113.7–114.9 °C. IR (KBr, cm−1) 3344br (NH), 1700s (CvO), (1H, s), 8.30 (3H, br s), 7.86 (1H, d, JH–F = 13.1 Hz), 7.50 (1H,
3
1653s (CvO), 1624s (CvO). 1H NMR (CDCl3, 400 MHz) δH 8.49 d, JH–F = 7.3 Hz), 4.50–4.49 (1H, m), 3.76–3.69 (4H, m), 3.72
4
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(3H, s), 3.69–3.67 (1H, m), 3.39–3.22 (4H, m), 2.80–2.78 (2H, δH 8.47 (1H, s), 7.64 (1H, d, JH–F = 12.2 Hz), 7.46 (1H, d,
m), 1.85–1.79 (2H, m), 1.77–1.68 (2H, m), 1.28–1.21 (2H, m), 4JH–F = 6.12 Hz), 4.87–4.84 (1H, br s), 3.89–3.76 (4H, br m),
1.10–1.09 (2H, m). m/z (ESI); 231 ([M + 2H]2+, 100%); HRMS 3.63–3.47 (2H, br m), 3.39–3.26 (3H, br m), 3.22–3.15 (2H, m),
(ESI) Calc. for C23H32N5O4F 230.6214. Found [M + 2H]2+, 2.79–2.44 (8H, m), 1.75–1.66 (2H, br m), 1.60–1.52 (2H, br m),
230.6214 (0 ppm error).
1.34–1.33 (2H, m), 1.08 (2H, br s). 13C NMR (D2O, 100 MHz) δC
Data for 6c. Molecular formula: C24H32N5O4F·2HCl, mole- 181.44 (CvO), 181.28 (CvO), 179.80 (CvO), 173.63 (CvO),
1
cular weight: 546.471 g mol−1. Yield = 0.235 g, 0.43 mmol, 173.39 (CvO), 172.84 (CvO), 154.12 (ipso-Ar, JC–F = 248 Hz),
1
2
98%. H NMR (d4-MeOH, 400 MHz) δH 8.69 (1H, s), 7.80 (1H, 148.19 (CH), 145.07 (ipso-Ar, d, JC–F = 10.7 Hz), 139.65
3
2
d, JH–F = 12.7 Hz), 7.52 (1H, br s), 4.62 (1H, br s), 3.96 (2H, br (ipso-Ar), 114.26 (ipso-Ar), 112.09 (CH, d, JC–F = 23.0 Hz),
s), 3.88 (3H, s), 3.82 (1H, br s), 3.75 (2H, br s), 3.47–3.35 (4H, 107.51 (CH), 76.05 (4°-C), 75.89 (4°-C), 50.71 (CH), 50.34 (CH2),
br m), 3.02 (2H, br s), 1.96 (2H, br s), 1.80 (2H, br s), 1.60 (2H, 46.34 (CH2), 45.52 (CH2), 45.03 (CH2), 43.01 (CH2), 39.96
br s), 1.42 (2H, br s), 1.18 (2H, br s). 13C NMR (d4-MeOH, (CH2), 39.73 (CH2), 39.15 (CH2), 36.14 (CH), 28.14 (CH2), 25.53
100 MHz) δC 174.45 (CvO), 168.71 (CvO), 166.68 (CvO), (CH2), 8.38 (CH2). m/z (ESI); 794 ([M + H]+, 100%); HRMS (ESI)
154.81 (ipso-Ar, d, 1JC–F = 249 Hz), 150.14 (CH), 146.12 (ipso-Ar, Calc. for C34H41N5O16 794.2527. Found [M + H]+, 794.2569
2
d, JC–F = 9.97 Hz), 140.07 (ipso-Ar), 122.29 (ipso-Ar), 112.85 (3.3 ppm error).
3
(CH, d, JC–F = 23.8 Hz), 109.12 (ipso-Ar), 107.45 (CH), 52.53
Data for 9b (S-ornCpf). Molecular formula: C34H35N5O16-
(CH3), 51.60 (CH), 51.14 (CH2), 50.56 (CH2), 46.55 (CH2), 43.25 FNa5, molecular weight: 903.621 g mol−1. Yield = 0.065 g,
(CH2), 40.41 (CH2), 36.93 (CH), 31.52 (CH2), 28.17 (CH2), 22.72 0.072 mmol, 17%; Rf 0.44 (1 : 1 MeCN : H2O); m.p. dec.
(CH2), 8.69 (CH2, C). m/z (ESI); 496 ([M + Na]+, 53%), 237 ([M + 213.5 °C. IR (KBr, cm−1) 3406s (OH), 2930w (CH), 1629s
H]2+, 100%); HRMS (ESI) Calc. for C24H32N5O4FNa 496.2331. (CvO), 1477m, 1448m, 1401m, 1337s (C–O), 1297m (C–O),
Found [M + Na]+, 496.2327 (1.0 ppm error).
1248s (C–O), 1145w, 1110w, 1026w. 1H NMR (D2O, 400 MHz)
3
Data for 6d. Molecular formula: C22H30N5O4F·2HCl, mole- δH 8.46 (1H, s), 7.64 (1H, d, JH–F = 12.2 Hz), 7.46 (1H, d,
cular weight: 520.428 g mol−1. Yield = 0.187 g, 0.36 mmol, 4JH–F = 6.12 Hz), 4.85–4.88 (1H, br s), 3.86–3.75 (4H, br m),
1
82%. H NMR (d6-DMSO, 400 MHz) δH 8.67 (1H, s), 8.42 (3H, 3.60–3.49 (2H, br m), 3.40–3.27 (3H, br m), 3.21–3.16 (2H, m),
br s), 8.21 (3H, br s), 7.83 (1H, d, 3JH–F = 13.18 Hz), 7.14 (1H, d, 2.81–2.45 (8H, m), 1.76–1.66 (2H, br m), 1.58–1.52 (2H, br m),
4JH–F = 7.32 Hz), 4.50–4.48 (1H, m), 4.45 (2H, br q, JH–H = 6.96 1.34–1.34 (2H, m), 1.06 (2H, br s). 13C NMR (D2O, 100 MHz) δC
3
Hz), 3.78–3.73 (4H, m), 3.73 (3H, s), 3.39–3.29 (4H, m), 181.56 (CvO), 181.29 (CvO), 179.78 (CvO), 173.61 (CvO),
1
2.80–2.79 (2H, m), 1.83–1.80 (2H, m), 1.77–1.67 (2H, m), 1.37 173.39 (CvO), 172.85 (CvO), 154.16 (ipso-Ar, JC–F = 248 Hz),
3
2
(3H, t, JH–H = 6.96 Hz). HRMS (ESI) Calc. for C22H31N5O4F 148.23 (CH), 145.05 (ipso-Ar, d, JC–F = 10.7 Hz), 139.67
448.2355. Found [M + H]+, 448.2369 (3.4 ppm error).
(ipso-Ar), 114.25 (ipso-Ar), 112.11 (CH, d, JC–F = 23.0 Hz),
2
Data for 8a. Molecular formula: C39H50N5O16F, molecular 107.52 (CH), 76.06 (4°-C), 75.89 (4°-C), 50.70 (CH), 50.36 (CH2),
weight: 863.837 g mol−1. Yield = 0.248 g, 0.29 mmol, 65%. 46.35 (CH2), 45.54 (CH2), 45.05 (CH2), 43.02 (CH2), 39.97
HRMS (ESI) Calc. for C39H51N5O16F 864.3309. Found [M + H]+, (CH2), 39.73 (CH2), 39.16 (CH2), 36.16 (CH), 28.16 (CH2), 25.54
864.3294 (1.6 ppm error); [M − MeOH]+, 832.3020, [M − 2 MeOH]+, (CH2), 8.38 (CH2). m/z (ESI); 794 ([M + H]+, 100%); HRMS (ESI)
800.2747.
Calc. for C34H41N5O16 794.2527. Found [M + H]+, 794.2568
Data for 8b. Molecular formula: C39H50N5O16F, molecular (3.3 ppm error).
weight: 863.837 g mol−1. Yield = 0.239 g, 0.28 mmol, 64%.
Data for 9c (S-lysCpf). Molecular formula: C35H37N5O16FNa5,
HRMS (ESI) Calc. for C39H51N5O16F 864.3309. Found [M + H]+, molecular weight: 917.648
g . Yield = 0.09 g,
mol−1
864.3297 (1.2 ppm error); [M − MeOH]+, 832.3023, [M − 2 MeOH]+, 0.098 mmol, 22%; m.p. dec. 124.8–126.7 °C. IR (KBr, cm−1
800.2749.
)
3403s (OH), 1653s (CvO), 1628s (CvO), 1587s (CvO). 1H
Data for 8c. Molecular formula: C40H52N5O16F, molecular NMR (D2O, 400 MHz) δH 8.43 (1H, s), 8.35–8.34 (1H, br m),
3
4
weight: 877.873 g mol−1. Yield = 0.281 g, 0.32 mmol, 72%. 7.59 (1H, d, JH–F = 12.9 Hz), 7.41 (1H, d, JH–F = 6.84 Hz),
HRMS (ESI) Calc. for C40H53N5O16F 878.3470. Found [M + H]+, 7.29–7.24 (1H, br m), 4.79 (1H, br s), 3.89–3.76 (4H, br m), 3.55
878.3467 (0.3 ppm error); [M − MeOH]+, 846.3221, [M − 2 MeOH]+, (1H, br s), 3.45–3.26 (4H, br m), 3.16–3.12 (2H, m), 2.76–2.44
814.3762.
(8H, m), 1.74–1.73 (2H, br m), 1.53–1.47 (2H, br m), 1.45–1.37
Data for 8d. Molecular formula: C38H50N5O16F, molecular (2H, br m), 1.33–1.31 (2H, m), 1.06–1.00 (2H, m). 13C NMR
weight: 851.826 g mol−1. Yield = 0.342 g, 0.40 mmol, 91.2%. (D2O, 100 MHz) δC 180.60 (CvO), 180.44 (CvO), 179.38
HRMS (ESI) Calc. for C38H51N5O16F 852.3309. Found [M + H]+, (CvO), 172.64 (CvO), 172.52 (CvO), 172.25 (CvO), 153.20
852.3275 (3.4 ppm error); [M − MeOH]+, 820.3020, [M − 2 MeOH]+, (ipso-Ar, 1JC–F = 249 Hz), 147.28 (CH), 144.14 (ipso-Ar, d, 2JC–F
=
788.2772.
10.7 Hz), 138.70 (ipso-Ar), 120.93 (ipso-Ar), 111.19 (CH, d,
Data for 9a (R-ornCpf). Molecular formula: C34H35N5O16- 2JC–F = 23.0 Hz), 106.57 (CH), 75.19 (4°-C), 75.02 (4°-C), 49.83
FNa5, molecular weight: 903.621 g mol−1. Yield = 0.050 g, (CH), 49.46 (CH2), 48.79 (CH2), 45.46 (CH2), 44.62 (CH2), 44.13
0.055 mmol, 13%; Rf 0.43 (1 : 1 MeCN : H2O); m.p. dec. (CH2), 42.12 (CH2), 39.15 (CH2), 35.25 (CH), 30.84 (CH2), 28.14
212.6–213.8 °C. IR (KBr, cm−1) 3406s (OH), 2932w (CH), 1628s (CH2), 22.40 (CH2), 7.50 (CH2). m/z (ESI); 830 ([M + Na]+,
(CvO), 1476m, 1448m, 1400m, 1337s (C–O), 1297m (C–O), 100%); HRMS (ESI) Calc. for C35H42N5O16FNa 830.2503. Found
1248s (C–O), 1144w, 1112w, 1025w. 1H NMR (D2O, 400 MHz) [M + Na]+, 830.2505 (0.1 ppm error).
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Data for 9d (S-ornNrf). Molecular formula: C33H35N5O16-
Organic & Biomolecular Chemistry
Notes and references
FNa5, molecular weight: 891.610 g mol−1. Yield = 0.11 g,
0.12 mmol, 14%; Rf 0.58 (1 : 1 MeCN : H2O); m.p. dec.
212.2–215.1 °C. IR (KBr, cm−1) 3413br (OH), 2974w (CH),
2937w (CH), 1627s (CvO), 1586s (CvO), 1491m, 1446m, 1401s,
1 J. M. Roosenberg, Y.-M. Lin, Y. Lu and M. J. Miller, Curr.
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7 U. Möllmann, L. Heinisch, A. Bauernfeind, T. Köhler and
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Chem., 1996, 35, 6429.
1
1245m (C–O), 1200w, 1142w, 1114w, 1089w, 1051w, 1025w. H
3
NMR (D2O, 400 MHz) δH 8.53 (1H, s), 7.88 (1H, d, JH–F
=
4
13.1 Hz), 7.14 (1H, d, JH–F = 6.40 Hz), 4.88–4.75 (1H, m), 4.42
(2H, br q, JH–H = 13.7, 6.72 Hz), 3.95–3.74 (4H, m), 3.40–3.31
3
(4H, m), 3.20–3.19 (2H, m), 2.75–2.42 (8H, m), 1.74–1.68 (2H,
3
m), 1.64–1.52 (2H, m), 1.47 (3H, t, JH–H = 7.32 Hz). 13C NMR
(D2O, 100 MHz) δC 181.27 (CvO), 179.80 (CvO), 179.12
(CvO), 176.32 (CvO), 173.65 (CvO), 173.41 (CvO), 172.87
1
(CvO), 172.39 (CvO), 154.09 (ipso-Ar, JC–F = 249 Hz), 148.03
2
(CH), 145.50 (ipso-Ar, d, JC–F = 10.7 Hz), 137.82 (ipso-Ar),
2
122.43 (ipso-Ar), 112.40 (CH, d, JC–F = 23.8 Hz), 106.77 (CH),
76.05 (4°-C), 75.89 (4°-C), 50.74 (CH2), 50.53 (CH2), 50.42 (CH),
46.34 (CH2), 45.52 (CH2), 45.02 (CH2), 43.06 (CH2), 39.73
(CH2), 29.46 (CH2), 25.54 (CH2), 14.60 (CH3). m/z (ESI); 781
9 S. Konetschny-Rapp, G. Jung, J. Meiwes and H. H. Zähner,
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([M + H]+, 46%); HRMS (ESI) Calc. for C33H41N5O16F 782.2527. 10 H. Drechsel and G. Winkelmann, BioMetals, 2005, 18, 75.
Found [M + H]+, 782.2506 (5.3 ppm error).
11 J. C. Grigg, J. D. Cooper, J. Cheung, D. E. Heinrichs and
Antimicrobial activity testing. Antimicrobial activity was
M. E. P. Murphy, J. Biol. Chem., 2010, 285, 11162.
assessed against a panel of 19 reference and clinical strains of 12 H. Drechsel, G. Jung and G. Winkelmann, BioMetals, 1992,
bacteria, representing 7 different species, obtained from the 5, 141.
culture collection of the Bradford Infection Group. A standard 13 S. R. Md-Saleh, E. C. Chilvers, K. G. Kerr, S. J. Milner,
disc diffusion assay was used, whereby the diameter of zones of
inhibition around discs impregnated with the test compounds
on lawns of each strain were measured. Bacteria were first har-
A. M. Snelling, J. P. Weber, G. H. Thomas, A.-K. Duhme-
Klair and A. Routledge, Bioorg. Med. Chem. Lett., 2009, 19,
1496.
vested from overnight agar plates and suspended in sterile 14 O. Keller, W. E. Keller, G. van Look and G. Wersin, Org.
saline to McFarland turbidity standard 0.5. Suspensions were Synth., 1985, 63, 160.
used to make confluent lawns of each strain on Iso-Sensitest 15 W. H. Miller, M. A. Seefeld, K. A. Newlander, I. N. Uzinskas,
agar (Oxoid Ltd). Sterile filter paper discs (Fischer Scientific,
0.6 cm diameter) were impregnated with 5 μg of ciprofloxacin
or norfloxacin stock or an equimolar amount of the respective
conjugate. Commercially available 5 μg ciprofloxacin discs
(Oxoid Ltd) were also used to determine ciprofloxacin suscepti-
W. J. Burgess, D. A. Heerding, C. C. K. Yuan, M. S. Head,
D. J. Payne, S. F. Rittenhouse, T. D. Moore, S. C. Pearson,
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3246.
bility, as defined by CLSI criteria. Discs were placed on the agar 16 G. Han, M. Tamaki and V. J. Hruby, J. Pept. Res., 2001, 58,
surface, and plates incubated for 18–20 h at 37 °C, and the dia-
meter of zones of inhibition were then recorded.
338.
17 K. Hirota, H. Kitagawa, M. Shimamura and S. Ohmori,
DNA gyrase assay. Assays were performed using the DNA
Chem. Lett., 1980, 191.
gyrase supercoiling assay from Inspiralis according to the man- 18 H. Guo, S. A. Naser, G. Ghobrial and O. Phanstiel, J. Med.
ufacturer’s instructions and based on the method of Maxwell Chem., 2002, 45, 2056.
et al. (2006).23 Success of the assay was absolutely dependent 19 H. Koga, A. Itoh, S. Murayama, S. Suzue and T. Irikura,
on the use of a gel electrophoresis apparatus that had never J. Med. Chem., 1980, 23, 1358.
come into contact with ethidium bromide as clearly extremely 20 Clinical and Laboratory Standards Institute, Performance
small amounts of this compound can lead to total failure of the
assay to resolve the different super-coiled forms of the plasmid.
standards for antimicrobial susceptibility testing; 16th infor-
mational supplement, CLSI M100-S16, Clinical and Labora-
tory Standards Institute, Wayne, PA, 2006.
21 C. Ji and M. J. Miller, Bioorg. Med. Chem., 2012, 20, 3828.
22 S. Noël, V. Gasser, B. Pesset, F. Hoegy, D. Rognan,
I. J. Schalk and G. L. A. Mislin, Org. Biomol. Chem., 2011, 9,
8288.
23 A. Maxwell, N. P. Burton and N. O’Hagan, Nucleic Acids
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Acknowledgements
We thank the University of York for financial support and
Leeanne Rayner for technical assistance with the antimicrobial
assays.
Org. Biomol. Chem.
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