Studies on the antimicrobial properties of N-acylated ciprofloxacins

Article abstract of DOI:10.1016/j.bmcl.2012.05.026

Fluoroquinolone antibiotics have been a mainstay in the treatment of bacterial diseases. The most notable representative, ciprofloxacin, possesses potent antimicrobial activity; however, a rise in resistance to this agent necessitates development of novel derivatives to prolong the clinical lifespan of these antibiotics. Herein we have synthesized and analyzed the antimicrobial properties of a library of N-acylated ciprofloxacin analogues. We find that these compounds are broadly effective against Gram-positive and Gram-negative bacteria, with many proving more effective than the parental drug, and several possessing MICs ≤1.0 μg/ml against methicillin-resistant Staphylococcus aureus and Bartonella species. An analysis of spontaneous mutation frequencies reveals very low potential for resistance in MRSA compared to existing fluoroquinolones. Mode of action profiling reveals that modification of the piperazinyl nitrogen by acylation does not alter the effect of these molecules towards their bacterial target. We also present evidence that these N-acylated compounds are highly effective at killing intracellular bacteria, suggesting the suitability of these antibiotics for therapeutic treatment.

Full text of DOI:10.1016/j.bmcl.2012.05.026

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6513–6520
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Studies on the antimicrobial properties of N-acylated ciprofloxacins
Ryan Cormier a, , Whittney N. Burda b, , Lacey Harrington b, Jordan Edlinger a, Karthik M. Kodigepalli b,
b,
John Thomas c, Rebecca Kapolka c, Glen Roma c, Burt E. Anderson c, Edward Turos a, , Lindsey N. Shaw
⇑
⇑
a Center for Molecular Diversity in Drug Design, Discovery, and Delivery, Department of Chemistry, University of South Florida, Tampa, FL 33620, USA
b Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, Tampa, FL 33620, USA
c Department of Molecular Medicine, College of Medicine, University of South Florida, Tampa, FL 33620, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Fluoroquinolone antibiotics have been a mainstay in the treatment of bacterial diseases. The most nota-
ble representative, ciprofloxacin, possesses potent antimicrobial activity; however, a rise in resistance to
this agent necessitates development of novel derivatives to prolong the clinical lifespan of these antibi-
otics. Herein we have synthesized and analyzed the antimicrobial properties of a library of N-acylated
ciprofloxacin analogues. We find that these compounds are broadly effective against Gram-positive
and Gram-negative bacteria, with many proving more effective than the parental drug, and several pos-
Received 6 March 2012
Revised 30 April 2012
Accepted 8 May 2012
Available online 1 July 2012
Keywords:
sessing MICs 61.0 lg/ml against methicillin-resistant Staphylococcus aureus and Bartonella species. An
N-Acyl ciprofloxacins
Staphylococcus aureus
MRSA
Antimicrobial activity
Antibiotic resistance
analysis of spontaneous mutation frequencies reveals very low potential for resistance in MRSA com-
pared to existing fluoroquinolones. Mode of action profiling reveals that modification of the piperazinyl
nitrogen by acylation does not alter the effect of these molecules towards their bacterial target. We also
present evidence that these N-acylated compounds are highly effective at killing intracellular bacteria,
suggesting the suitability of these antibiotics for therapeutic treatment.
Ó 2012 Published by Elsevier Ltd.
Over the last 50 years, the fluoroquinolone antibiotics have
been a mainstay in the treatment of bacterial diseases. The most
notable member of this family, ciprofloxacin, possesses potent
antimicrobial activity against a broad spectrum of Gram-negative
and Gram-positive pathogens and, despite recent evidence of bac-
terial strains having fluoroquinolone resistance, remains one of the
foremost lines of defense against pathogenic bacteria.1 Broadly,
quinolone-based antibiotics inhibit bacterial DNA replication by
interfering with the ability of DNA gyrase and topoisomerase IV
to reseal nicked DNA prior to strand passage.2 Acquired resistance
to the fluoroquinolones is mediated through chromosomal muta-
tions in bacterial genes encoding these enzymes at specific do-
mains, known as the Quinolone Resistance-Determining Regions
(QRDR). Resistance can also occur via mutations that affect import
and/or export of the drug, via non-specific efflux mechanisms.2a,3
More recently there have been reports of plasmid-mediated resis-
tance mechanisms, including the quinolone resistance proteins
Qnr, Aac(60) Ib-cr and QepA.4
opportunity to accomplish this would be through attachment of
lipophilic acyl residues to the nitrogen of the piperazinyl ring. Re-
cent studies have indicated that increased bulkiness of alkyl sub-
stituents at this site enhances protection from efflux exporter
proteins, and decreases bacterial drug-resistance.5 The antimicro-
bial properties of a small number of N-acylated ciprofloxacins have
previously been described in the patent literature.6 As of yet, there
have been no detailed investigations into how well these (and re-
lated) analogues may function against drug-resistant bacteria, or
whether there might be perturbations to their mode of action as
antibacterial agents. In this report we describe the first such stud-
ies on N-acylated ciprofloxacin analogues and their microbiological
properties against representative pathogenic bacteria, including
multi-drug resistant strains, and investigate whether they act by
interfering with the existing and known mechanisms of action
for this class of compounds.
A focused library of 18 lipophilic, N-acylated ciprofloxacin
derivatives 2a–r were synthesized using the published procedure
of Azema, by treating ciprofloxacin with the requisite acyl chloride
or acid anhydride7 in the presence of triethylamine at room tem-
perature (Fig. 1). The desired N-acylated products were obtained
in 32–96% yields after chromatography, and suitably characterized
by proton and carbon NMR spectroscopy.
In an attempt to overcome pathways of bacterial drug-resis-
tance, we set out to explore ways to enhance bioactivity of cipro-
floxacin. Given the mode of action and well-characterized
structure–activity requirements of this drug, we viewed the best
The antimicrobial properties of these N-acylated ciprofloxacin
derivatives were evaluated against several key Gram-positive and
Gram-negative bacteria by Kirby-Bauer disk diffusion assay,
⇑
Corresponding authors.
E-mail address: eturos@shell.cas.usf.edu (E. Turos).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2012 Published by Elsevier Ltd.
http://dx.doi.org/10.1016/j.bmcl.2012.05.026

6514
R. Cormier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6513–6520
O
O
N
O
O
N
F
F
HO
HO
RCOCl or RC(O)OC(O)R
N
N
Et3N, CH2Cl2
rt
N
R
NH
O
2a-r
Figure 1. Synthesis of N-acylated ciprofloxacin derivatives 2a–r.
Table 1
Table 2
Results of Kirby-Bauer testing of N-acylated ciprofloxacins against MRSA USA 100
Minimum inhibitory concentrations of N-acyl ciprofloxacins 2a–r against MSSA and
MRSA
Compound
R
MRSA (CBD-635)
Compound
R
MSSA
(SH1000)
MRSA (ATCC
43300)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
Methyl
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
24
20
6
30
36
30
6
22
34
26
6
30
33
31
35
28
22
6
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
Methyl
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
10
40
100+
7.5
20
25
25
10
10
10
100+
100+
1
25
100+
25
10
10
100+
100+
1
Nonyl
CH(CH2CH3)CH2CH2CH2CH3
CH(CH2CH2CH3)2
CH(CH2CH3)2
CH(CH3)CH2CH2CH3
C(CH3)2CH2CH3
CH2CH(CH3)CH2(CH3)3
CH2CH(CH3)2
C(CH3)3
Nonyl
CH(CH2CH3)CH2CH2CH2CH3 10
CH(CH2CH2CH3)2
CH(CH2CH3)2
CH(CH3)CH2CH2CH3
C(CH3)2CH2CH3
CH2CH(CH3)CH2(CH3)3
CH2CH(CH3)2
C(CH3)3
10
100+
10
5
25
100+
7.5
25
1
1
2q
2r
100+
100+
100+
100+
15
Phenyl
Ciprofloxacin
6
2q
2r
Phenyl
Data is shown in millimeters and represents the average diameter of the zone of
inhibition from three independent experiments. Each assay was performed with
Ciprofloxacin
10
50
lg of drug per disk. For those compounds that displayed no activity, a zone of
Data shown is in ug/ml of antibiotic compound, tested in triplicate and averaged.
6 mm is shown, which corresponds to the diameter of the disk.
Table 3
Spontaneous mutation frequencies for selected N-acylated ciprofloxacin analogues
determination of the minimum inhibitory concentration (MIC),
DNA gyrase activity assay, spontaneous mutation frequency assay,
and intracellular viability assay.
The N-acyl ciprofloxacins were tested for in vitro bioactivity
against three separate Gram-positive bacteria, including Staphylo-
coccus aureus, Bacillus anthracis and Enterococcus faecalis. For the
staphylococci, methicillin-susceptible Staphylococcus aureus
(MSSA) and methicillin-resistant Staphylococcus aureus (MRSA)
strains were examined for comparison.
Several strains of S. aureus were used in testing the N-acyl
ciprofloxacins 2a–r. The clinical isolate CBD-635 (MRSA, USA100)
was selected for initial disk diffusion assays, and ATCC strain
43300 (MRSA), the laboratory strain SH1000 (MSSA) and CBD-635
(MRSA) were employed for the minimum inhibitory concen-
tration assays.8
Disk diffusion assays were performed in triplicate, as previously
described, with the average zones of bacterial growth inhibition of
each compound shown in Table 1.8 All but four (2c, 2g, 2k, and 2r)
of the N-acylated ciprofloxacin derivatives we tested had greater
anti-MRSA activity than ciprofloxacin, with the most active of the
analogs being N-hexanoyl derivative 2e.
Compound
1.0ꢀ
1.5ꢀ
2.0ꢀ
2.5ꢀ
2a
2b
2i
2m
90
ND
Lawn
Lawn
ND
41
101
ND
Lawn
4
0
0
ND
Lawn
7
Lawn
0
551
Ciprofloxacin
ND
ND
Numbers in the upper row of the table refer to fold increase of the MIC. Lawn refers
to a complete covering of the plate with bacterial cells, ND = Not Determined. The
values indicate the total colonies obtained for at least 3 independent replicates per
compound.
averaged MIC values shown in Table 2. Ciprofloxacin was used as
a positive control. Against the MSSA strain, derivatives 2a, 2d,
2h, 2i, 2j, 2k, 2m, 2n, and 2q were all as active as ciprofloxacin,
while 2d, 2n, and 2q showed slightly better activity. With regards
to the MRSA strain, 2a, 2d, 2h, 2i, 2l, 2m, and 2n gave MIC values
lower than that of ciprofloxacin. Curiously, compounds 2d, 2l, 2m,
and 2n all showed enhanced bioactivity towards the MRSA than
the MSSA.
Given the ease with which S. aureus develops resistance to anti-
microbial agents, we undertook spontaneous mutation frequency
assays with selected compounds from our library (Table 3).8 For this
we chose three representatives (2a, 2i and 2m), which each had
The minimum inhibitory concentrations of the N-acyl ciproflox-
acins were evaluated against Staphylococcus aureus SH1000 and the
multidrug-resistant MRSA strain CBD-635 according to previous
published procedures.8 None of the derivatives exhibited discern-
ible inhibitory activity toward CBD-635 below a concentration of
MICs of 10
lg/ml in our MSSA assay, and 2b, which had an MIC of
40 g/ml. In addition, we also included ciprofloxacin as a control
l
100 lg/ml (data not shown). Consequently, we elected to use an-
agent for these studies. As such, agar containing 2a, 2i and 2m at
1ꢀ-, 1.5ꢀ-, 2.0ꢀ- and 2.5ꢀ MIC was prepared, alongside media con-
taining ciprofloxacin at 2.5ꢀ MIC. When inoculated with overnight
cultures of MSSA we found that all four concentrations of 2i
other more common MRSA strain (ATCC 43300), which shows only
limited resistance to antibiotics beyond b-lactam compounds. All
the antimicrobial assays were performed in triplicate, with the

R. Cormier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6513–6520
6515
Table 4
Table 5
Kirby-Bauer assay of N-acylated ciprofloxacins 2a–r against B. anthracis and E. faecalis
Kirby-Bauer assay of N-acylated ciprofloxacins 2a–r against B. henselae and B.
quintana
Compound
R
B. anthracis
E. faecalis
(Sterne)
(DS16)
Compound
R
B. henselae
B. quintana
(ATCC 49882) (ATCC VR358)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
Methyl
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
89
86
86
80
77
71
65
55
6
48
46
40
39
38
36
29
23
6
28
26
22
33
45
25
32
44
6
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
Methyl
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
52
56
34
38
31
28
16
6
6
58
29
35
30
20
14
11
11
6
Nonyl
CH(CH2CH3)CH2CH2CH2CH3 65
Nonyl
10
CH(CH2CH2CH3)2
CH(CH2CH3)2
CH(CH3)CH2CH2CH3
C(CH3)2CH2CH3
CH2CH(CH3)CH2(CH3)3
CH2CH(CH3)2
C(CH3)3
72
81
67
57
80
70
80
49
42
CH(CH2CH3)CH2CH2CH2CH3 12
CH(CH2CH2CH3)2
CH(CH2CH3)2
CH(CH3)CH2CH2CH3
C(CH3)2CH2CH3
CH2CH(CH3)CH2(CH3)3
CH2CH(CH3)2
C(CH3)3
14
33
37
41
15
62
40
21
56
6
17
24
27
7
36
33
7
2q
2r
Phenyl
2q
2r
Ciprofloxacin
31
Phenyl
Ciprofloxacin
6
Data is shown in millimeters and represents the average diameter of the zone of
inhibition from three independent experiments. Each assay was performed with
50 lg of drug per disk. For those compounds that displayed no zone of growth
Data is shown in millimeters and represents the average diameter of the zone of
inhibition from three independent experiments. Each assay was performed with
inhibition, a value of 6 mm is shown, which corresponds to the diameter of the disk.
20 lg of drug per disk. For those compounds that displayed no activity, a zone of
6 mm is shown, which corresponds to the diameter of the disk.
produced lawns of growth, suggesting that resistance is readily
developed for this compound. We also obtained a lawn of growth
for 2m at 1ꢀ MIC; however, we observed significantly fewer colo-
nies at higher concentrations, with none even being detectable at
2.5ꢀ MIC. From all tests, we isolated eleven 2m-resistant colonies
from a total inoculum of 1.2 ꢀ 10ꢁ10. This yielded a spontaneous
mutation rate of 1.08 ꢀ 10ꢁ9 for this agent. Testing with compound
2a provided resistant colonies for each of the concentrations tested,
apart from 2.5ꢀ MIC, which failed to produce growth. In total we
isolated 232 colonies for 2a, from a combined inoculum of
1.7 ꢀ 1010, yielding a mutation rate of 7.3 ꢀ 10ꢁ7. Given the ele-
vated MIC of 2b, we chose the single, and commonly employed,
concentration of 2.5ꢀ MIC for analysis. Despite repeating this assay
six times, using a combined bacterial inoculum of 3.67 ꢀ 1010, we
were unable to find any mutant colonies. In contrast to these find-
ings, when using a combined inoculum of 5.38 ꢀ 108 on agar con-
taining 2.5ꢀ MIC of ciprofloxacin, we obtained 551 colonies from
five individual tests. This results in a spontaneous mutation fre-
quency of 1.02 ꢀ 10ꢁ6 for the parent drug. As such, this is a more
than 71-fold increase in mutation frequency when compared to
2a, and a more than 1000-fold increase when compared to 2m. This
significance is further heightened by the observation that no resis-
tance to either 2a or 2m was observed at the 2.5ꢀ MIC concentra-
tions used for ciprofloxacin.
N-acyl ciprofloxacins 2a–r were also tested against B. anthracis
(Sterne)9 and E. faecalis (DS16)10 using the previously described
Kirby–Bauer disk diffusion assay (Table 4). The N-acyl ciprofloxac-
ins performed well in this assay, with only one compound (2r) fail-
ing to surpass ciprofloxacin in bioactivity against B. anthracis.
Derivatives 2a, 2b, 2c, 2d, 2e, 2f, 2m, 2n, 2p, and 2q all fared better
than the positive control against E. faecalis. For both bacteria,
bioactivity dropped as the length or lipophilicity of the acyl chain
increased.
N-Acyl ciprofloxacin compounds 2a–r were also tested and
found to be effective against Bartonella and Escherichia coli,
clinically-significant Gram-negative microbes. Disk diffusion as-
says were evaluated against four species of Bartonella, including
B. henselae (ATCC 49882)11, B. quintana (ATCC VR358), B. elizabet-
hae (F9251)12, and B. vinsonii (ATCC VR152). Minimum inhibitory
concentration assays were performed only with the B. henselae
strain. All assays were run in triplicate. While the N-acyl
ciprofloxacins showed inhibitory activity against the four Barton-
ella species tested (Tables 5 and 6), most produced diminished
growth inhibition zone sizes compared to ciprofloxacin. A general
relationship between lipophilicity and activity was observed with
the more hydrophobic compounds yielding smaller growth inhibi-
tion zones.
The minimum inhibitory concentrations of the N-acyl ciproflox-
acins were and evaluated against B. henselae. Ciprofloxacin was
used as a positive control with the averaged MIC values shown
in Table 7. Compounds 2b, 2c, and 2n were as active as ciproflox-
acin, whereas 2p and 2r displayed higher activity than the control.
Table 6
Kirby-Bauer assay of N-acylated ciprofloxacins 2a–r against B. elizabethae and B.
vinsonii
Compound
R
B. elizabethae B. vinsonii
(F9251)
(ATCC VR152)
2a
2b
2c
2d
2e
2f
2g
2h
Methyl
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
58
57
42
35
30
20
14
12
9
ND
60
44
57
21
22
15
14
9
2i
Nonyl
2j
2k
2l
2m
2n
2o
2p
2q
CH(CH2CH3)CH2CH2CH2CH3 12
15
6
CH(CH2CH2CH3)2
CH(CH2CH3)2
CH(CH3)CH2CH2CH3
C(CH3)2CH2CH3
CH2CH(CH3)CH2(CH3)3
CH2CH(CH3)2
C(CH3)3
6
26
32
32
8
37
36
13
58
25
30
33
10
40
36
12
58
2r
Phenyl
Ciprofloxacin
Data is shown in millimeters and represents the average diameter of the zone of
inhibition from three independent experiments. Each assay was performed with
20
lg of drug per disk. ND = Not Determined. For those compounds that displayed
no activity, a zone of 6 mm is shown, which corresponds to the diameter of the disk.

6516
R. Cormier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6513–6520
E. coli.15 Several of the derivatives proved to be more potent than
ciprofloxacin. 2a, 2b, 2c, 2d, 2f, and 2q all yielded larger zones than
the positive control as shown in Table 8.
A major requirement for any potential antibiotic targeted to-
wards bacterial species is that it must have a prokaryotic target
that is selective and distinct from any eukaryotic counterpart.
Therefore, in order to assess the relative toxicity of our library to-
wards eukaryotic cells we repeated our disk diffusion studies using
Saccharomyces cerevisiae. Encouragingly, none of the compounds
tested were toxic to this organism at concentrations used for the
antimicrobial testing (data not shown).
As fluoroquinolones target enzymes that mediate DNA supercoil-
ing, we employed a classic biochemical assay to measure DNA gyr-
ase activity in the presence of select compounds.16 All compounds
tested (2a, 2b, 2q, 2e and 2o) exhibited a clear ability to inhibit
the supercoiling activity of purified E. coli DNA gyrase when tested
Table 7
Minimum inhibitory concentration assay of N-acylated ciprofloxacins 2a–r against B.
henselae
Compound
R
B.henselae (ATCC 49882)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
Methyl
Ethyl
Propyl
Butyl
Pentyl
Hexyl
Heptyl
Octyl
ND
0.5
0.5
5
5
10
10
10
5
ND
5
Nonyl
CH(CH2CH3)CH2CH2CH2CH3
CH(CH2CH2CH3)2
CH(CH2CH3)2
CH(CH3)CH2CH2CH3
C(CH3)2CH2CH3
CH2CH(CH3)CH2(CH3)3
CH2CH(CH3)2
C(CH3)3
5
0.8
0.5
1
0.2
ND
0.2
0.5–113
with pUC19 at concentrations ranging from 1.0 to 100 lg/ml
(Fig. 3). Of note, tests with compound 2e appeared to demonstrate
minimalsupercoiling of the plasmid at even very low concentrations
of the antibiotic (1.0 lg/ml). Thus, all five test compounds have
2q
2r
Phenyl
Ciprofloxacin
inhibitory activity against DNA gyrase, indicating that N-acylation
of ciprofloxacin does not abrogate gyrase inhibitory capabilities.
In addition to this biochemical approach, we also undertook
DNA sequencing analyses of our spontaneously generated 2a and
2m MSSA mutants. Accordingly, we sequenced the Quinolone
Resistance-Determining Regions (QRDR) of the genes encoding
DNA gyrase (gyrA and gyrB) and topoisomerase IV (grlA and grlB)17
from representative mutant isolates. We also conducted this anal-
ysis in parallel on our parental MSSA strain, and used the data de-
rived to identify mutations arising in these regions. The sequence
data for all mutants revealed identical mutations in each strain
in the gyrA and grlA genes, regardless of compound used to gener-
ate them. Specifically, S84L and E88 K mutations were observed in
gyrA, and S80Y and E84G mutations in grlA. Interestingly, whilst
the former three mutations are well characterized for ciprofloxacin
resistance in S. aureus, the latter has only been documented rarely.
Indeed, E84G mutation of grlA is more commonly associated in S.
aureus with resistance to derivatives of ciprofloxacin, such as
Determined by agar dilution, data shown is in ug/ml of antibiotic compound, tested
in triplicate and averaged. ND = not determined.
In order to assess the activity of representative compounds
against the facultative intracellular bacterium B. henselae, and bio-
availability inside the cell, we performed cell infection assays using
immortalized microvascular cell line HMEC-1 as described previ-
ously.14 After extracellular bacteria were killed with gentamicin,
infected cells were incubated for 96 h with select test compounds.
At a concentration of 1.0 lg/ml, all compounds tested were effec-
tive at reducing the number of intracellular bacteria to levels
610% of those found in infected cells exposed only to solvent con-
trols (Fig. 2). At 0.1 lg/ml, the number of surviving bacteria found
in cell lysates was higher, ranging from 32% for compound 2p, to
54% for compounds 2a and 2n.
The Kirby-Bauer assay was used to determine the N-acyl
ciprofloxacins antimicrobial assay against the D5H
a strain of
Figure 2. Assay for intracellular antimicrobial activity of selected N-acylated ciprofloxacin compounds against Bartonella henselae. HMEC cells were infected with B. henselae
for 4 h, before being incubated with select compounds at the concentration specified for 96 h. Values are shown as percentage of colony forming units in comparison to
control, which contained media only. Error bars are shown as SD.

R. Cormier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6513–6520
6517
Table 8
in different ways. For examples, in the case of the Gram-negative
organisms E. coli and Bartonella species, there is a general trend
of decreasing bioactivity for the compounds as the acyl chain
length increases, which coincides with increasing lipophilicity.
The calculated values for logP, a measurement of a compounds
lipophilic character, are provided in Table 9, and plotted out versus
anti-Bartonella bioactivity in Fig. 4.
This trend is most clear for Bartonella elizabethae, but is also
seen with other Bartonella species, and, to a lesser extent, E. coli
and B. anthracis (data not shown). Conversely, there is no apparent
lipophilicity-activity correlation for MRSA and only a very weak
trend for E. faecalis (data not shown). It is interesting that Azema
and colleagues reported an analogous observation for ciprofloxacin
derivatives bearing lipophilic N-side chains in their antitumor
properties against five human cancer cell lines.7
Disk diffusion assay of N-acylated ciprofloxacins 2a–r against E. coli
Compound
R
E. coli (D5Ha)
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
Methyl
Ethyl
Propyl
Butyl
pentyl
Hexyl
Heptyl
Octyl
60
57
48
47
30
45
6
30
6
6
´
Nonyl
CH(CH2CH3)CH2CH2CH2CH3
CH(CH2CH2CH3)2
CH(CH2CH3)2
CH(CH3)CH2CH2CH3
C(CH3)2CH2CH3
CH2CH(CH3)CH2(CH3)3
CH2CH(CH3)2
C(CH3)3
6
39
33
38
6
36
51
6
Using a biochemical assay, we were able to demonstrate that
select representatives of our library efficiently inhibited the ability
of purified E. coli DNA gyrase to super-coil the plasmid pUC19.
Furthermore, when analyzing strains of S. aureus having developed
resistance to compounds 2a and 2m, we observed point mutations
in both gyrA and grlA genes of DNA gyrase, and topoisomerase IV,
respectively. As such, it would appear that modification of the
piperazinyl nitrogen by acylation does not alter the manner in
which these molecules act toward their bacterial target. With re-
gards to the potential for resistance, we demonstrate that, whilst
spontaneous mutation was readily obtained for one of the com-
pounds (2i), resistance to others was a far less frequent occurrence.
Specifically, we obtained a cumulative mutation frequency of
7.3 ꢀ 10ꢁ7 for 2a and 1.2 ꢀ 10ꢁ10 for 2m. In contrast we herein
2q
2r
Phenyl
Ciprofloxacin
43
Data is shown in millimeters and represents the average diameter of the zone of
inhibition from three independent experiments. Each assay was performed with
50 lg of drug per disk. For those compounds that displayed no activity, a zone of
6 mm is shown, which corresponds to the diameter of the disk
trovafloxacin, norfloxacin and besofloxacin.18 No mutations were
observed in the QRDR of gyrB or grlB of the mutant strains.
Prior structure–activity studies on the fluoroquinolones starting
in the 1970’s have enabled substantial improvements in their po-
tency, spectrum of activity, and in vivo efficacy. Essentially every
site on the quinolone framework has been chemically derivatized
and evaluated for antibacterial activities, leading to a well-defined
understanding of the optimal groups for each site in terms of elec-
trostatics, size, and shape. Included in this list are the C6 fluoro
substituent, and the C7 piperazinyl side chain found in ciprofloxa-
cin, and its related structural analogues. Data from the present
study indicates that N-acylation of ciprofloxacin not only affects,
but can improve, the antibacterial activity of this drug against a
variety of bacterial species. When compared to the parental com-
pound, we observed a general increase in efficacy for the derivative
compounds. Indeed, it appears that N-acylation of ciprofloxacin
significantly improves antibacterial activity towards Gram-positive
organisms. Specifically, with regards to the Kirby-Bauer assays,
only four derivatives proved less effective than ciprofloxacin when
tested against the MRSA strain CBD-635 (2c, 2g, 2k and 2r), eight
against E. faecalis (2g, 2h, 2i, 2j, 2k, 2l, 2o and 2r), and only one
(2i) had decreased activity compared to ciprofloxacin against B.
anthracis. Additionally, a number of compounds showed improve-
ment in activity over ciprofloxacin against Gram-negative organ-
isms, when tested against E. coli and B. quintana.
show
a
spontaneous mutation frequency of 1.02 ꢀ 10ꢁ6 for
ciprofloxacin at 2.5ꢀ MIC. As such, this is a more than 71-fold in-
crease in resistance frequency when compared to 2a, and a more
than 1000-fold increase when compared to 2m. This clearly dem-
onstrates that the N-acylated ciprofloxacin derivatives in our li-
brary have vastly lower mutation frequencies than for
ciprofloxacin, which is of particular importance given that cipro-
floxacin is rarely used in treating S. aureus infections due to rela-
tively high resistance rates.19 By and large, the mutations
obtained within our resistant strains were classical for this type
of antimicrobial agent. Specifically, the S84L and E88 K mutations
in gyrA, and S80Y mutation in grlA have previously been reported
for ciprofloxacin. With regards to the E84G mutation of grlB, this
is far less common, and is more frequently associated with resis-
tance to trovafloxacin, norfloxacin and besofloxacin.18 As such,
the more favorable mutation frequency, coupled with an unusual
collection of point mutations required to achieve resistance, sug-
gests the potential suitability of these compounds for treating S.
aureus infections.
The N-acylated ciprofloxacin derivatives were even more effec-
tive against Gram-negative bacteria, and have lower MICs than
those for S. aureus. Most Gram-negative bacteria, and Bartonella
Interestingly, N-acylated ciprofloxacins appear to alter the
growth and survival of Gram-positive and Gram-negative bacteria
Figure 3. The effects of selected N-acyl ciprofloxacin compounds on DNA Gyrase activity. Relaxed circular (R) pUC19 DNA was incubated in the presence of E. coli DNA gyrase
and decreasing concentrations (100 g/mL, 50 g/mL, 10 g/mL and 1.0 g/ml) of select compounds. Gyrase conversion of pUC19 to its supercoiled (S) form was inhibited by
increasing concentrations of each compound. Control samples without compound (C), in the absence (ꢁ) or presence (+) of DNA gyrase, are also shown.
l
l
l
l

6518
R. Cormier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6513–6520
Table 9
gest the physiological relevance and suitability of these com-
pounds as a potential treatment option.22
N-Acyl ciprofloxacins in increasing order of lipophilicity, as determined by their
calculated logP values (ChemDraw, version 7.0)
Compound
R
Calculated logP
Disclaimer
2a
2b
2c
2q
2p
2d
2n
2r
Methyl
Ethyl
Propyl
C(CH3)3
CH2CH(CH3)2
Butyl
C(CH3)2CH2CH3
Phenyl
CH(CH2CH3)2
CH(CH3)CH2CH2CH3
Pentyl
0.17
0.7
Any opinions, findings, and conclusions or recommendations
expressed in this publication are those of the author(s) and do
not necessarily reflect the views of the DARPA.
1.23
1.41
1.63
1.76
1.96
1.99
2.07
2.07
2.29
2.81
3.12
3.12
3.34
3.48
3.87
4.4
Acknowledgments
2l
This study was supported in part by grant 1R01AI080626-01A2
(LNS) from the National Institute of Allergies and Infectious
Diseases, and by the DARPA (BA and ET) under Award No.
HR0011-08-1-0087.
2m
2e
2f
Hexyl
2j
CH(CH2CH3)CH2CH2CH2CH3
CH(CH2CH2CH3)2
Heptyl
CH2CH(CH3)CH2(CH3)3
Octyl
2k
2g
2o
2h
2i
References and notes
Nonyl
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R.; Andrews, J. M.; Edwards, L. J. Antimicrob. Agents Chemother. 1983, 23, 559.
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Sugino, A.; Peebles, C. L.; Kreuzer, K. N.; Cozzarelli, N. R. Proc. Nat’l. Acad. Sci.
1977, 74, 4767.
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Sawada, T.; Izumiya, H.; Watanabe, H. Gut 2010, 2, 17; (b) Gay, K.; Robicsek, A.;
Strahilevitz, J.; Park, C. H.; Jacoby, G.; Barrett, T. J.; Medalla, F.; Chiller, T. M.;
Hooper, D. C. Clin. Infect. Dis. 2006, 43(3), 297; (c) Martínez-Martínez, L.;
Pascual, A.; Jacoby, G. A. Lancet 1998, 351, 797; (d) Sjolund-Karlsson, M.; Howie,
R.; Rickert, R.; Krueger, A.; Tran, T. T.; Zhao, S.; Ball, T.; Haro, J.; Pecic, G.; Joyce,
K.; Fedorka-Cray, P. J.; Whichard, J. M.; McDermott, P. F. Emerg. Infect. Dis. 2010,
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Antimicrob. Agents Chemother. 2000, 44, 798; (b) Davies, T. A.; Kelly, L. M.;
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M.; Machado, D.; Martins, M.; Melo-Cristino, J.; Amaral, L.; Couto, I. BMC
Microbiology 2011, 11, 241.
6. Petersen, U. G., K.; Kiihle, E.; Zeiler, H.; Metzger, K. G. US patent 4559341, 1984.
7. Azéma, J.; Guidetti, B.; Dewelle, J.; Le Calve, B.; Mijatovic, T.; Korolyov, A.;
Vaysse, J.; Malet-Martino, M.; Martino, R.; Kiss, R. Bioorg. Med. Chem. 2009, 17,
5396.
8. Burda, W.; Fields, K.; Gill, J.; Burt, R.; Shepherd, M.; Zhang, X.; Shaw, L.;
European, J. Clin. Microbiol. Infect. Dis. 2012, 327.
Figure 4. Antimicrobial activity of N-acyl ciprofloxacin derivatives against Gram-
negative bacteria is inversely proportional to their lipophilicity. Values shown are
the zones of inhibition against Bartonella elizibethae (in mm), versus the compound
listed in order of increasing logP.
9. Sterne, M. Onderstepoort. J. Vet. Sci. Anim. Ind. 1937, 9, 49.
10. Gawron-Burke, C.; Clewell, D. B. Nature 1982, 300, 281.
11. Regnery, R. L.; Anderson, B. E.; Clarridge, J. E.; Rodriguez-Barradas, M. C.; Jones,
D. C.; Carr, J. H. J. Clin. Microbiol. 1992, 30, 265.
12. Daly, J. S.; Worthington, M. G.; Brenner, D. J.; Moss, C. W.; Hollis, D. G.; Weyant,
R. S.; Steigerwalt, A. G.; Weaver, R. E.; Daneshvar, M. I.; O’Connor, S. P. J. Clin.
Microbiol. 1993, 31, 872.
13. Dörbecker, C.; Sander, A.; Oberle, K.; Schülin-Casonato, T. J. Antimicrob.
Chemother. 2006, 58, 784.
14. (a) Ades, E. W.; Candal, F. J.; Swerlick, R. A.; George, V. G.; Summers, S.; Bosse,
D. C.; Lawley, T. J. J. Investig. Dermatol. 1992, 99, 683; (b) Ives, T. J.; Marston, E.
L.; Regnery, R. L.; Butts, J. D. Int. J. Antimicrob. Agents 2001, 18, 217.
15. Sambrook, J.; Fritsch, E. F.; Maniatis, T. T. Molecular Cloning A Laboratory
Manual, second ed.; Cold Spring Harbor Laboratory Press: Cold Spring Harbor,
New York, 1989.
16. Maxwell, A.; Burton, N. P.; O’Hagan, N. Nucl. Acids Res. 2006, 34, e104.
17. Horii, T.; Suzuki, Y.; Monji, A.; Morita, M.; Muramatsu, H.; Kondo, Y.; Doi, M.;
Takeshita, A.; Kanno, T.; Maekawa, M. Diag. Microbiol. Infect. Dis. 2003, 46, 139.
18. (a) Fitzgibbon, J. E.; John, J. F.; Delucia, J. L.; Dubin, D. T. Antimicrob. Agents
Chemother. 1998, 42, 2122; (b) Haas, W.; Pillar, C. M.; Hesje, C. K.; Sanfilippo, C.
M.; Morris, T. W. J. Antimicrob. Chemother. 2010, 65, 1441; (c) Kosmidis, C.;
DeMarco, C. E.; Frempong-Manso, E.; Seo, S. M.; Kaatz, G. W. Int. J. Antimicrob.
Agents 2010, 36, 222.
species in particular, are sensitive to quinolones, however, newer
drugs have been reported to exhibit greater activity than ciproflox-
acin.20 In addition, both naturally occurring mutations and labora-
tory generated mutations in the QRDR of gyrA have been reported
and associated with fluoroquinolone resistance in Bartonella spe-
cies.21 Accordingly, the development of novel quinolone deriva-
tives, such as those presented in this study, is desirable. This
contention is enhanced by the finding that select members of our
library were able to efficiently kill intracellular B. henselae. Previ-
ously, it was reported that levofloxacin (MIC = 0.84
better than ciprofloxacin (MIC = 15.2 g/ml), sparfloxacin
(MIC = 6.4 g/ml) and ofloxacin (MIC = 5.6 g/ml) at killing intra-
cellular B. henselae in infected Vero cells,
lg/ml) was
l
l
l
14b
suggesting that the
newer antibiotic variants may possess better intracellular activity.
As such, the observation that the N-acylated compounds from our
library induced 50–70% killing of intracellular bacteria at 0.1
lg/
ml, and almost complete killing at 1.0 g/ml, suggests very real
l
19. Blumber, H. M.; Rimland, D.; Carroll, D. J.; Terry, P.; Wachsmuth, I. K. J. Infect.
Dis. 1991, 163, 1279.
20. Rolain, J. M.; Brouqui, P.; Koehler, J. E.; Maguina, C.; Dolan, M. J.; Raoult, D.
Antimicrob. Agents Chemother. 2004, 48, 1921.
21. (a) Angelakis, E.; Biswas, S.; Taylor, C.; Raoult, D.; Rolain, J.-M. J. Antimicrob.
Chemother. 2008, 61, 1252; (b) Minnick, M. F.; Wilson, Z. R.; Smitherman, L. S.;
Samuels, D. S. Antimicrob. Agents Chemother. 2003, 47, 383.
22. Experimental procedures and data on the N-acyl ciprofloxacins.
Bacterial strains and growth conditions
enhancements in activity for these derivatives over existing
fluoroquinolones. Indeed, the development of fluoroquinolones
with enhanced intracellular activity is not only critical for treating
infections caused by Bartonella species, but also infections caused
by obligate intracellular bacteria as well. These findings, coupled
with the lack of toxicity to the eukaryote S. cerevisiae, further sug-

R. Cormier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6513–6520
6519
E. coli, S. aureus, E. faecalis, B. anthracis and S. cerevisiae strains were grown as
General methods for the synthesis of N-acyl ciprofloxacins 2a–r
Method A: Ciprofloxacin (500 mg, 1.5 mmol) and triethylamine (300 ll,
described previously.8 Bartonella strains were cultured on chocolate agar
prepared from heart infusion agar base supplemented with 5% bovine
hemoglobin at 37 °C in 5% CO2.
2 mmol) were stirred in 20 mL of methylene chloride at 0 °C for 15 min. The
desired acyl chloride (2.25 mmol) was added dropwise. The suspension was
allowed to stir at room temperature until a clear solution was observed. To this
solution, hexane was added drop wise until a white precipitate formed. The
precipitate was then filtered off and dried. If further purification was needed,
the desired compound was isolated via flash chromatography using 20%
methanol in dichloromethane as the eluent.
Disk diffusion sensitivity assays
Disk diffusion assays for S. aureus, B. anthracis, E. faecalis, E. coli and S. cerevisiae
were performed as described previously.8 Owing to the fastidious nature of
Bartonella spp., standardized susceptibility testing guidelines (CLSI or EUCAST)
are not available. As such, these assays were performed as described
previously, with the following modifications.13 Twenty microliters of
relevant antibiotics, at a concentration of 1 mg/ml, were spotted onto the
center of 6 mm paper disks (BBL) on a sheet of aluminum foil, in a biological
safety cabinet. Disks were allowed to dry for 20 min, and then stored in a
sealed bag with desiccant at 4 °C. Growth from 4 day old plates was
resuspended in 1.0 ml sterile Heart Infusion Broth, and turbidity was
adjusted to a McFarland 2.0 by visual inspection. The bacterial suspension
was spread over the surface of a chocolate agar plate using a swab. The
inoculum was allowed to dry into the agar in a biological safety cabinet for
15 min. Disks were then placed in the center of plates, which were inverted
and incubated at 37 °C in a 5% CO2 incubator for one week. For all organisms,
the zone of inhibition was measured by recording the diameter, to the nearest
mm, for each disk.
Method B: Ciprofloxacin (500 mg, 1.5 mmol) and triethylamine (300 lL,
2 mmol) were stirred in 20 mL of methylene chloride at 0 °C for 15 min. The
desired acid anhydride (3 mmol) was added dropwise. The suspension was
allowed to stir at room temperature until a clear solution was observed. To this
solution, hexane was added drop wise until a white precipitate formed. The
precipitate was then filtered off and dried. If further purification was needed,
the desired compound was isolated via flash chromatography using 20%
methanol in dichloromethane as the eluent.
7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (2a). Obtained 460 mg (81%) as an off-white solid. Melting
POINT: >260 °C. 1H NMR (400 MHz, CDCl3)
d ppm 8.70 (s, 1H) 7.96 (d,
J = 12.8 Hz, 1H) 7.34 (d, J = 6.6 Hz, 1H) 3.77 (m, 4H) 3.53 (br s, 1H) 3.31 (m, 4H)
2.14 (s, 3H) 1.38 (d, J = 5.4 Hz, 2H) 1.18 (br s, 2H) 13C NMR (101 MHz, CDCl3) d
ppm 177.0 (d, J = 3.0 Hz), 169.1, 166.8, 153.6 (d, J = 250.0 Hz), 147.5, 145.4 (d,
J = 10.7 Hz), 139.0, 120.2 (d, J = 7.6 Hz), 112.5 (d, J = 23.0 Hz), 108.1, 50.1, 49.4,
46.0, 41.0, 35.3, 21.3, 8.2
Minimum inhibitory concentration determination
The minimum inhibitory concentration of compounds against MRSA and MSSA
strains was determined as described previously.8 MICs for Bartonella strains
were determined via agar dilution methods. Briefly, strains were tested for
7-(4-propionylpiperazin-1-yl)-1-Cyclopropyl-6-fluoro-4-oxo-1,4-
growth on chocolate agar plates containing antibiotics at 10.0
ml, and 0.1 g/ml. Compounds inhibiting growth at 61.0 g/ml were further
tested to determine the more precise MIC using 2-fold dilutions at and below
1.0 g/ml. Agar plates containing DMSO (without compound) as a control were
l
g/ml, 1.0
l
g/
dihydroquinoline-3-carboxylic acid (2b). Obtained 530 mg (91%) as an off-white
solid. Melting Point: >260 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.69 (s, 1H) 7.95
(d, J = 12.8 Hz, 1H) 7.33 (d, J = 6.6 Hz, 1H) 3.77 (m, 4H) 3.53 (br s, 1H) 3.31 (m,
4H) 2.39 (q, J = 7.4 Hz, 2H) 1.37 (d, J = 5.0 Hz, 2H) 1.17 (m, 5H) 13C NMR
l
l
l
prepared at the highest dilution to assess any antibacterial activity associated
with the solvent. Growth from four day old chocolate agar plates was collected
for each Bartonella strain tested. The growth was suspended into 0.5 ml of
(101 MHz, CDCl3) d ppm 176.9 (d, J = 3.1 Hz), 172.4, 166.7, 153.6 (d,
J = 251.6 Hz), 147.4, 145.4 (d, J = 10.9 Hz), 138.9, 112.4 (d, J = 23.2 Hz), 108.1,
105.0 (d, J = 3 Hz), 50.1, 49.3, 45.1, 41.1, 35.3, 26.4, 9.3, 8.2
sterile Heart Infusion broth. The turbidity was adjusted to
a
McFarland
7-(4-Butyryl-piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-
standard of 2.0 by visual comparison to turbidity standards. Twenty five
microliter of each bacterial suspension was spotted onto plates containing
varying concentrations of drug. Chocolate agar plates containing no antibiotics
were used as controls to confirm viability. Inoculation drops were allowed to
briefly dry into the agar. Plates were inverted and incubated at 37 °C with a 5%
CO2 atmosphere for 7 days. Growth was recorded as + or ꢁ for each strain on
duplicate plates.
dihydroquinoline-3-carboxylic acid (2c). Obtained 589 mg (97%) as an off-
white solid. Melting Point: >260 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.73 (s,
1H) 8.00 (d, J = 12.9 Hz, 1H) 7.34 (d, J = 7.0 Hz, 1H) 3.79 (m, 4H) 3.53 (m, 1H)
3.30 (m, 4H) 2.35 (m, 2H) 1.69 (m, 2H) 1.38 (d, J = 6.6 Hz, 2H) 1.18 (d, J = 2.7 Hz,
2H) 0.98 (t, J = 7.4 Hz, 3H) 13C NMR (101 MHz, CDCl3) d ppm 177.0, 171.6,
166.8, 154.8 (d, J = 251.5 Hz), 147.5, 145.4 (d, J = 10.7 Hz), 138.97, 120.3 (d,
J = 9.1 Hz) 112.6 (d, J = 24.5 Hz), 108.2, 105.0 (d, J = 3.0 Hz), 50.3, 49.4, 41.0,
35.2, 35.1, 18.6, 13.9, 8.2
Derivation of spontaneous mutation frequencies
TSB agar (TSA) was prepared containing N-acyl ciprofloxacin derivatives 2a, 2i
or 2m at concentrations equivalent to 1ꢀ, 1.5ꢀ, 2.0ꢀ and 2.5ꢀ the
experimentally-determined MIC for MSSA. For 2b, TSA plates were prepared
at a concentration equivalent to 2.5ꢀ MIC for MSSA. Overnight broth cultures
of MSSA were prepared as described previously8, with 1 ml aliquots extracted,
and cells harvested by centrifugation. Supernatants were removed, and pellets
1-Cyclopropyl-6-fluoro-4-oxo-7-(4-pentanoyl-piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylic acid (2d). Obtained 423 mg (68%) as an off-white
solid. Melting Point: >260 °C 1H NMR (400 MHz, CDCl3) d ppm 8.56 (br s, 1H)
7.79 (d, J = 12.0 Hz, 1H) 7.29 (d, J = 7.4 Hz, 1H) 3.76 (m, 4H) 3.54 (m, 1H) 3.30
(m, 4H) 2.34 (t, J = 8.0 Hz, 2H) 1.59 (quin, J = 7.5 Hz, 2H) 1.34 (m, 4H) 1.16 (m,
2H) 0.89 (t, J = 7.2 Hz, 3H) 13C NMR (101 MHz, CDCl3) d ppm 176.7, 171.8,
166.5, 153.5 (d, J = 250.1 Hz), 147.3, 145.3 (d, J = 11.0 Hz), 138.9, 119.6, 112.0
(d, J = 23.4 Hz), 107.7, 105.0 (d, J = 4.1 Hz), 50.1, 49.3, 45.3, 41.0, 35.3, 32.9,
27.3, 22.5, 13.8, 8.1
1-Cyclopropyl-6-fluoro-7-(4-hexanoyl-piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (2e). Obtained 531 mg (83%) as an off-
white solid. Melting Point: 204–206 °C. 1H NMR (400 MHz, CDCl3) d ppm 0.80
(t, J = 6.3 Hz, 3H) 1.10 (m, 2H) 1.28 (m, 6H) 1.55 (br s, 2H) 2.29 (t, J = 7.4 Hz, 2H)
3.23 (m, 4H) 3.52 (br s, 1H) 3.72 (m, 4H) 7.24 (br s, 1H) 7.66 (d, J = 12.9 Hz, 1H)
8.46 (br s, 1H) 13C NMR (101 MHz, CDCl3) d ppm 176.5, 171.8, 166.5, 153.3 (d,
J = 251.6 Hz), 147.2, 145.3 (d, J = 7.6 Hz), 138.8, 119.4 (d, J = 9.9 Hz), 111.7 (d,
J = 20.7 Hz), 107.5, 105.0 (d, J = 3.2 Hz), 49.9, 49.3, 45.2, 41.0, 35.3, 33.1, 24.8,
22.3, 13.8, 8.1
resuspended in 100 ll of fresh TSB. These preparations were then used to
inoculate the N-acyl ciprofloxacin-containing agar, and spread using sterile
glass beads. The colony forming units (cfu) per ml of the inoculating culture
was determined via serial dilution into TSA containing no antibiotic compound.
Spontaneous mutation frequencies were calculated by dividing the number of
colonies obtained by the total bacterial load inoculated.
Sequence analysis of quinolone binding domains for spontaneously resistant strains
DNA was extracted from spontaneously resistant MSSA mutants using
a
DNeasy kit (Qiagen), according to the manufacturer’s instructions. Samples
were subject to DNA sequencing reactions (MWG) using primers specific for
the Quinolone Resistance-Determining Regions (QRDR) of the gyrAB and grlAB
genes of S. aureus, as described previously by Horii et al.17
Assay for intracellular activity against Bartonella henselae
1-Cyclopropyl-6-fluoro-7-(4-heptanoyl-piperazin-1-yl)-4-oxo-1,4-
The HMEC-1 human microvascular endothelial cell line was maintained in
dihydroquinoline-3-carboxylic acid (2f). Obtained 560 mg (84%) as an off-white
solid. Melting Point: 162–164 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.66 (s, 1H)
7.92 (d, J = 12.9 Hz, 1H) 7.32 (d, J = 7.0 Hz, 1H) 3.77 (m, 4H) 3.53 (tt, J = 7.0,
3.7 Hz, 1H) 3.31 (m, 4H) 2.35 (t, J = 8.0 Hz, 2H) 1.63 (quin, J = 7.5 Hz, 2H) 1.33
(m, 8H) 1.17 (m, 2H) 0.86 (t, J = 8.0 Hz, 3H) 13C NMR (101 MHz, CDCl3) d ppm
176.9, 171.8, 166.7, 153.5 (d, J = 252.6 Hz), 147.4, 145.4 (d, J = 9.3 Hz), 138.9,
120.0, 112.3 (d, J = 24.9 Hz), 108.0, 105.0 (d, J = 3.4 Hz), 50.1, 49.3, 45.3, 41.0,
35.2, 33.2, 31.5, 29.1, 25.2, 22.5, 14.0, 8.2
MCDB131 medium supplemented with 10% FBS, 5%
and 1
g/ml hydrocortisone.1 HMEC-1 were infected with the Houston-1 strain
of B. henselae at an MOI of 100 for 4 h as previously described.22 After infection,
g/ml)
L-glutamine, 10 ng/ml EGF,
l
the cells were washed 2ꢀ with PBS, then treated with gentamicin (200
l
for 1 h to kill extracellular adherent bacteria. Infected cells were washed as
before and media with test antibiotics were added at concentrations of 0.1 and
1.0 lg/ml. After addition of test antibiotics, infected cells were incubated for
96 h. Following incubation, the antibiotics were removed, the infected cells
were washed as before, and lysed with 0.1% saponin. Lysates were plated on
chocolate agar and incubated for 7 days. After incubation, the CFU/ml were
counted to determine the number of viable bacteria.
DNA gyrase activity assay
The activity of select compounds against DNA gyrase was tested using relaxed
circular pUC19 DNA in the presence of E. coli DNA gyrase and antibiotics at
1-Cyclopropyl-6-fluoro-7-(4-octanoyl-piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (2g). Obtained 656 mg (95%) as an off-white
solid. Melting Point: 154–156 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.70 (s, 1H)
7.96 (d, J = 12.9 Hz, 1H) 7.33 (d, J = 7.0 Hz, 1H) 3.77 (m, 4H) 3.53 (br s, 1H) 3.31
(m, 4H) 2.36 (t, J = 7.8 Hz, 2H) 1.65 (s, 2H) 1.32 (m, 12H) 0.86 (t, J = 7.0 Hz, 3H)
13C NMR (101 MHz, CDCl3) d ppm 177.0, 171.8, 166.7, 153.6 (d, J = 250.3 Hz),
147.4, 145.4 (d, J = 10.7 Hz), 138.9, 120.1, 112.5 (d, J = 23.0 Hz), 108.1, 105.0 (d,
J = 3.4 Hz), 50.2, 49.4, 45.3, 41.0, 35.2, 33.2, 31.6, 29.4, 29.0, 25.2, 22.5, 14.0, 8.2
1-Cyclopropyl-6-fluoro-7-(4-nonanoyl-piperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (2h). Obtained 693 mg (96%) as an off-white
solid. Melting Point: 136–142 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.73 (s, 1H)
8.00 (d, J = 12.9 Hz, 1H) 7.34 (d, J = 7.0 Hz, 1H) 3.80 (m, 4H) 3.52 (m, 1H) 3.30
(m, 4H) 2.36 (t, J = 7.4 Hz, 2H) 1.64 (m, 2H) 1.28 (m, 14H) 0.86 (t, J = 7.0 Hz, 3H)
13C NMR (101 MHz, CDCl3) d ppm 177.0, 171.8, 166.7, 153.5 (d, J = 248.7 Hz),
147.5, 145.4 (d, J = 10.8 Hz), 138.9, 120.3, 112.6 (d, J = 21.6 Hz), 108.2, 105.0 (d,
J = 2.9 Hz), 50.3, 49.4, 45.3, 41.0, 35.2, 33.2, 31.7, 29.4, 29.3, 29.1, 25.2, 22.6,
14.0, 8.2
concentrations of 1.0, 5.0, 10, and 25 lg/ml. Samples were incubated at 37 °C
for 1 h then analyzed by gel electrophoresis to quantify the amount of relaxed
and supercoiled DNA, as previously described.16
Synthetic procedures
All the chemicals used for the synthesis of the N-acylated ciprofloxacins were
purchased from Aldrich Chemical Company and used without further
purification. Thin layer chromatography was performed using Silica Gel 60
F
254 purchased from EMD Chemicals. A UVG-11 Minera light lamp was used to
visualize the TLC plates. The NMR spectra were recorded in deuterated
chloroform using an Inova 400 MHz instrument.

6520
R. Cormier et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6513–6520
1-Cyclopropyl-7-(4-decanoyl-piperazin-1-yl)-6-fluoro-4-oxo-1,4-
1-Cyclopropyl-7-[4-(2,2-dimethyl-butyryl)-piperazin-1-yl]-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (2n). Obtained 343 mg (53%) as an off-white
solid. Melting Point: 182–184 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.77 (s, 1H)
8.05 (d, J = 12.5 Hz, 1H) 7.34 (d, J = 7.8 Hz, 1H) 3.88 (m, 4H) 3.51 (m, 1H) 3.30
(m, 4H) 1.66 (q, J = 8.0 Hz, 2H) 1.39 (m, 2H) 1.27 (m, 6H) 1.19 (m, 2H) 0.91 (t,
J = 7.8 Hz, 3H) 13C NMR (101 MHz, CDCl3) d 177.1, 175.8, 166.9, 153.6 (d,
J = 251.6 Hz), 147.6, 145.4 (d, J = 10.7 Hz), 139.0, 138.9, 120.4, 112.7 (d,
J = 23.0 Hz), 108.3, 104.9, 50.0, 49.9, 44.7, 43.0, 35.3, 33.3, 26.5, 9.5, 8.3
1-Cyclopropyl-6-fluoro-4-oxo-7-[4-(3,5,5-trimethyl-hexanoyl)-piperazin-1-yl]-
1,4-dihydroquinoline-3-carboxylic acid (2o). Obtained 386 mg (54%) as an off-
white solid. Melting Point: 204–206 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.7 (s,
1H) 7.9 (d, J = 12.5 Hz, 1H) 7.3 (d, J = 6.3 Hz, 1H) 3.8 (m, 4H) 3.5 (m, 1H) 3.3 (m,
4H) 2.3 (m, 2H) 2.1 (m, 1H) 1.4 (d, J = 6.3 Hz, 2H) 1.2 (m, 4H) 1.0 (d, J = 6.3 Hz,
3H) 0.9 (m, 9H) 13C NMR (101 MHz, CDCl3) d 176.9, 171.2, 166.7, 153.5 (d,
J = 251.6 Hz), 147.4, 145.4 (d, J = 10.7 Hz), 139.0, 120.0, 112.3 (d, J = 23.0 Hz),
108.0, 105.0 (d, J = 3.0 Hz), 50.8, 50.3, 49.4, 45.5, 42.5, 41.0, 35.3, 31.1, 30.0,
27.1, 22.9, 8.2
1-Cyclopropyl-7-[4-(2,2-dimethyl-propionyl)-piperazin-1-yl]-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (2p). Obtained 462 mg (68%) as an off-white
solid. Melting Point: >260 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.7 (s, 1H) 7.9
(d, J = 12.9 Hz, 1H) 7.3 (d, J = 7.0 Hz, 1H) 3.8 (m, 4H) 3.5 (br s, 1H) 3.3 (m, 4H)
2.3 (d, J = 7.0 Hz, 2H) 2.1 (dt, J = 13.5, 6.5 Hz, 1H) 1.4 (d, J = 6.6 Hz, 2H) 1.2 (br s,
2H) 1.0 (d, J = 6.6 Hz, 6H) 13C NMR (101 MHz, CDCl3) d ppm 177.0, 171.1, 166.7,
153.5 (d, J = 250.0 Hz), 147.5, 145.4 (d, J = 10.7 Hz), 139.0, 120.1, 112.5 (d,
J = 24.6 Hz), 108.1, 105.1 (d, J = 3.0 Hz), 50.3, 49.5, 45.6, 42.0, 41.0, 35.3, 25.7,
22.5, 8.2
1-Cyclopropyl-7-[4-(2,2-dimethyl-propionyl)-piperazin-1-yl]-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (2q). Obtained 530 mg (85%) as an off-white
solid. Melting Point: >260 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.70 (s, 1H) 7.96
(d, J = 12.9 Hz, 1H) 7.33 (d, J = 7.0 Hz, 1H) 3.87 (m, 4H) 3.52 (m, 1H) 3.31 (m,
4H) 1.38 (m, 2H) 1.30 (s, 9H)1.19 (m, 2H) 13C NMR (101 MHz, CDCl3) d 177.0,
176.6, 166.8, 153.6 (d, J = 250.0 Hz), 147.5, 145.4 (d, J = 10.7 Hz), 139.0, 120.1,
112.5 (d, J = 24.6 Hz), 108.1, 105.0 (d, J = 3.0 Hz), 49.9, 49.8, 44.8, 38.7, 35.3,
28.4, 8.2
7-(4-Benzoyl-piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydro-
quinoline-3carboxylic acid (2r). Obtained 572 mg (87%) as an off-white solid.
Melting point: >260 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.8 (s, 1H) 8.0 (d,
J = 12.5 Hz, 1H) 7.5 (m, 6H) 3.9 (m, 4H) 3.6 (br s, 1H) 3.4 (m, 4H) 1.4 (d,
J = 6.6 Hz, 2H) 1.2 (br s, 2H) 13C NMR (101 MHz, CDCl3) d 176.0, 153.9 (d,
J = 253.1 Hz), 148.1, 145.9 (d, J = 9.2 Hz), 139.4, 132.9, 131.1, 128.9, 127.1,
119.1, 116.2, 113.4, 112.4 (d, J = 24.5 Hz), 106.9, 105.3 (d, J = 3.1 Hz), 50.1, 49.0,
47.8, 42.5, 36.1, 8.2.
dihydroquinoline-3-carboxylic acid (2i). Obtained 233 mg (32%) as an off-
white solid. Melting Point: 130–136 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.63
(s, 1H) 7.88 (d, J = 5.1 Hz, 1H) 7.31 (d, J = 6.6 Hz, 1H) 3.76 (m, 4H) 3.53 (br s, 1H)
3.30 (m, 4H) 2.35 (t, J = 7.6 Hz, 2H) 1.62 (m, 2H) 1.29 (m, 16H) 0.84 (dd, J = 7.0,
5.5 Hz, 3H) 13C NMR (101 MHz, CDCl3) d ppm 176.8, 171.9, 166.7, 153.4 (d,
J = 251.6 Hz), 147.4, 145.4 (d, J = 9.2 Hz), 138.9, 119.9, 112.3 (d, J = 23.0 Hz),
107.9, 105.1 (d, J = 3.1 Hz), 50.2, 49.4, 45.4, 41.1, 35.3, 33.2, 31.8, 29.3, 25.3,
22.6, 14.1, 8.2
1-Cyclopropyl-7-[4-(2-ethyl-hexanoyl)-piperazin-1-yl]-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (2j). Obtained 646 mg (93%) as an off-white
solid. Melting Point: 138–150 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.61 (s, 1H)
7.84 (d, J = 13.3 Hz, 1H) 7.30 (d, J = 7.0 Hz, 1H) 3.86 (m, 4H) 3.53 (br s, 1H) 3.30
(m, 4H) 2.58 (m, 1H) 1.64 (m, 2H) 1.34 (m, 10H) 0.85 (m, 5H) 13C NMR
(101 MHz, CDCl3)
d ppm 176.8 (d, J = 3.1 Hz),174.9, 166.7, 153.5 (d,
J = 251.6 Hz), 147.4, 145.3 (d, J = 10.7 Hz), 138.9, 119.8 (d, J = 7.7 Hz), 112.2
(d, J = 23.0 Hz), 107.9, 50.4, 49.6, 45.4, 42.5, 41.2, 35.3, 32.3, 29.8, 25.9, 22.8,
14.0, 12.1, 8.2
1-Cyclopropyl-6-fluoro-4-oxo-7-[4-(2-propyl-pentanoyl)-piperazin-1-yl]-1,4-
dihydroquinoline-3-carboxylic acid (2k). Obtained 331 mg (48%) as an off-white
solid. Melting Point: 178–179 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.54 (s, 1H)
7.74 (d, J = 12.9 Hz, 1H) 7.27 (d, J = 4.7 Hz, 1H) 3.79 (m, 4H) 3.53 (br s, 1H) 3.28
(m, 4H) 2.65 (m, 1H) 1.59 (m, 2H) 1.25 (m, 10H) 0.83 (t, J = 7.2 Hz, 6H) 13C NMR
(101 MHz, CDCl3) d ppm 176.7, 175.0, 166.7, 153.4 (d, J = 250.1 Hz), 147.3,
145.3 (d, J = 10.7 Hz), 138.9, 119.6 (d, J = 7.7 Hz), 112.0 (d, J = 27.6 Hz), 107.7,
105.0 (d, J = 3.0 Hz), 50.3, 49.5, 45.4, 41.2, 40.4, 35.4, 35.1, 20.8, 14.1, 8.1
1-Cyclopropyl-7-[4-(2-ethyl-butyryl)-piperazin-1-yl]-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (2l). Obtained 389 mg (58%) as an off-white
solid. Melting Point: 248–254 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.68 (s, 1H)
7.94 (d, J = 12.9 Hz, 1H) 7.33 (d, J = 7.0 Hz, 1H) 3.85 (m, 4H) 3.53 (dd, J = 7.0,
3.5 Hz, 1H) 3.31 (m, 4H) 2.54 (tt, J = 8.2, 5.3 Hz, 1H) 1.66 (m, 2H) 1.50 (m, 2H)
1.38 (q, J = 6.5 Hz, 2H) 1.18 (m, 2H) 0.88 (t, J = 7.4 Hz, 6H) 13C NMR (101 MHz,
CDCl3) d ppm 176.9, 174.6, 166.8, 153.5 (d, J = 251.6 Hz), 147.5, 145.4 (d,
J = 10.7 Hz), 139.0, 120.1, 112.4 (d, J = 24.6 Hz), 108.1, 105.0 (d, J = 3.0 Hz), 50.5,
49.6, 45.4, 44.1, 41.3, 35.3, 25.5, 12.1, 8.2
1-Cyclopropyl-6-fluoro-7-[4-(2-methyl-pentanoyl)-piperazin-1-yl]-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (2m). Obtained 504 mg (78%) as an off-white
solid. Melting Point: 182–184 °C. 1H NMR (400 MHz, CDCl3) d ppm 8.7 (s, 1H)
8.0 (d, J = 13.0 Hz, 1H) 7.3 (d, J = 7.1 Hz, 1H) 3.8 (m, 4H) 3.5 (br s, 1H) 3.3 (d,
J = 16.7 Hz, 4H) 2.7 (m, 1H) 1.3 (m, 8H) 1.1 (d, J = 6.8 Hz, 3H) 0.9 (t, J = 6.8 Hz,
3H) 13C NMR (101 MHz, CDCl3) d 176.9, 175.4, 166.8, 153.5 (d, J = 253.1 Hz),
147.5, 145.4 (d, J = 10.7 Hz), 139.0, 120.0, 112.4 (d, J = 23.0 Hz) 108, 108.0,
105.0 (d, J = 3.1 Hz), 50.4, 49.5, 45.3, 41.2, 36.2, 35.1, 20.6, 17.5, 14.1, 8.2