Effect of N-1/C-8 ring fusion and C-7 ring structure on fluoroquinolone lethality

Article abstract of DOI:10.1128/AAC.01054-10

Quinolones rapidly kill bacteria by two mechanisms, one that requires protein synthesis and one that does not. The latter, which is measured as lethal action in the presence of the protein synthesis inhibitor chloramphenicol, is enhanced by N-1 cyclopropyl and C-8 methoxy substituents, as seen with the highly lethal compound PD161144. In some compounds, such as levofloxacin, the N-1 and C-8 substituents are fused. To assess the effect of ring fusion on killing, structural derivatives of levofloxacin and PD161144 differing at C-7 were synthesized and examined with Escherichia coli. A fused-ring derivative of PD161144 exhibited a striking absence of lethal activity in the presence of chloramphenicol. In general, ring fusion had little effect on lethal activity when protein synthesis was allowed, but fusion reduced lethal activity in the absence of protein synthesis to extents that depended on the C-7 ring structure. Additional fused-ring fluoroquinolones, pazufloxacin, marbofloxacin, and rufloxacin, also exhibited reduced activity in the presence of chloramphenicol. Energy minimization modeling revealed that steric interactions of the trans-oriented N-1 cyclopropyl and C-8 methoxy moieties skew the quinolone core, rigidly orient these groups perpendicular to core rings, and restrict the rotational freedom of C-7 rings. These features were not observed with fused-ring derivatives. Remarkably, structural effects on quinolone lethality were not explained by the recently described X-ray crystal structures of fluoroquinolone-topoisomerase IV-DNA complexes, suggesting the existence of an additional drug-binding state. Copyright

Full text of DOI:10.1128/AAC.01054-10

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2010, p. 5214–5221
0066-4804/10/$12.00 doi:10.1128/AAC.01054-10
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 54, No. 12
Effect of N-1/C-8 Ring Fusion and C-7 Ring Structure on
Fluoroquinolone Lethalityᰔ
Muhammad Malik,2 Kevin R. Marks,1 Heidi A. Schwanz,1 Nadezhda German,1
Karl Drlica,2,3 and Robert J. Kerns1*
Division of Medicinal and Natural Products Chemistry, University of Iowa, Iowa City, Iowa 522421; Public Health Research Institute,
New Jersey Medical School, 225 Warren Street, Newark, New Jersey 071032; and Department of Microbiology and
Molecular Genetics, New Jersey Medical School, 225 Warren Street, Newark, New Jersey 071033
Received 30 July 2010/Returned for modification 26 August 2010/Accepted 9 September 2010
Quinolones rapidly kill bacteria by two mechanisms, one that requires protein synthesis and one that does
not. The latter, which is measured as lethal action in the presence of the protein synthesis inhibitor chlor-
amphenicol, is enhanced by N-1 cyclopropyl and C-8 methoxy substituents, as seen with the highly lethal
compound PD161144. In some compounds, such as levofloxacin, the N-1 and C-8 substituents are fused. To
assess the effect of ring fusion on killing, structural derivatives of levofloxacin and PD161144 differing at C-7
were synthesized and examined with Escherichia coli. A fused-ring derivative of PD161144 exhibited a striking
absence of lethal activity in the presence of chloramphenicol. In general, ring fusion had little effect on lethal
activity when protein synthesis was allowed, but fusion reduced lethal activity in the absence of protein
synthesis to extents that depended on the C-7 ring structure. Additional fused-ring fluoroquinolones, pazu-
floxacin, marbofloxacin, and rufloxacin, also exhibited reduced activity in the presence of chloramphenicol.
Energy minimization modeling revealed that steric interactions of the trans-oriented N-1 cyclopropyl and C-8
methoxy moieties skew the quinolone core, rigidly orient these groups perpendicular to core rings, and restrict
the rotational freedom of C-7 rings. These features were not observed with fused-ring derivatives. Remarkably,
structural effects on quinolone lethality were not explained by the recently described X-ray crystal structures
of fluoroquinolone-topoisomerase IV-DNA complexes, suggesting the existence of an additional drug-binding
state.
Fluoroquinolones are broad-spectrum antimicrobials that
trap DNA gyrase and DNA topoisomerase IV on DNA. For-
mation of a ternary drug-enzyme-DNA complex, which is re-
versed by removal of quinolone (5, 23), blocks DNA replica-
tion, RNA synthesis, and bacterial growth (3, 5, 21). Events
subsequent to complex formation are irreversible and kill bac-
teria. Rapid cell death, which correlates with chromosome
fragmentation, occurs by two pathways, one that requires pro-
tein synthesis and one that does not (reviewed in reference 4).
The former pathway also involves production of toxic hydroxyl
radical, since agents that inhibit hydroxyl radical accumulation
block the lethal action of quinolones, such as oxolinic acid (25).
The latter pathway does not require hydroxyl radical accumu-
lation for cell death (25). We have suggested that this pathway
involves drug-induced destabilization of ternary complexes
that leads to chromosome fragmentation, because fragmenta-
tion occurs when gyrase and fluoroquinolone are added to
isolated nucleoids (20). How quinolone structure contributes
to destabilization of ternary complexes is not understood.
Measurements of lethal action in the presence of chloram-
phenicol have identified several features of quinolone struc-
ture that contribute to chromosome fragmentation and cell
death in the absence of protein synthesis. One feature is the
substituent at position N-1. With Escherichia coli, ciprofloxacin
is lethal in the presence of chloramphenicol, while norfloxacin
is not (6, 18). The only structural difference between the two
compounds is the N-1 substituent, a cyclopropyl ring for cip-
rofloxacin and ethyl for norfloxacin. Another structural feature
is the presence of a methoxy group at C-8, which improves
lethal activity in the presence of chloramphenicol over com-
pounds lacking an 8-methoxy group, as observed with Staphy-
lococcus aureus (7, 27), E. coli (12–14, 17, 18), Mycobacterium
smegmatis (19), and Mycobacterium tuberculosis (16). The C-7
ring system is also important. With E. coli, a C-7 N-ethyl
piperazine renders the action of an N-1 cyclopropyl, C-8 me-
thoxy compound (PD161144) insensitive to chloramphenicol,
while the C-7 diazabicyclo ring of moxifloxacin affords this
insensitivity with mycobacteria (16, 20). Thus, structure-activity
relationships for killing in the absence of ongoing protein syn-
thesis are beginning to emerge.
The importance of N-1 cyclopropyl and C-8 methoxy groups
to quinolone lethality, particularly killing of cells in the ab-
sence of protein synthesis, raises questions about the lethality
of levofloxacin, a clinically important fluoroquinolone (10).
Levofloxacin can be envisioned as having its C-8 methoxy
group connected to a ring-opened N-1 cyclopropyl group, gen-
erating a rigid fused-ring structure in which the N-1 and C-8
groups cannot reposition or rotate relative to the quinolone
core during the formation of ternary complexes with gyrase
and DNA. Whether the rigid ring system that fuses N-1 and
C-8 structural motifs promotes or impedes the killing of chlor-
amphenicol-treated cells relative to growing cells can be de-
* Corresponding author. Mailing address: Division of Medicinal and
Natural Products Chemistry, University of Iowa, Iowa City, IA 52242.
Phone: (319) 335-8800. Fax: (319) 335-8766. E-mail: robert-kerns
@uiowa.edu.
ᰔ Published ahead of print on 20 September 2010.
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LETHALITY OF FLUOROQUINOLONES
5215
FIG. 1. Structures of fluoroquinolones used in the present work. C-7 variants of N-1 cyclopropyl, 8-methoxy fluoroquinolones and cognate
levofloxacin-like fused-ring derivatives were synthesized and compared to test structural requirements for killing. Gatifloxacin, levofloxacin,
marbofloxacin, moxifloxacin, pazufloxacin, and rufloxacin are commercially available products.
groups was achieved using established methods for nucleophilic aromatic sub-
stitution of a C-7 fluorine on commercially available fluoroquinolone interme-
diates. For N-1 cyclopropyl, C-8 methoxy derivatives (including PD161144,
PD135041, and PD161148), the commercially available secondary amines were
termined by comparison of derivatives that have the same C-7
ring systems.
In the present work, we prepared levofloxacin-like fused-
ring fluoroquinolone derivatives containing C-7 groups previ-
reacted with 1-cyclopropyl-6,7-difluoro-8-methoxy-4-oxo-3-quinolinecarboxylic
ously shown to affect killing in the absence of protein synthesis
when substituted on C-8 methoxy fluoroquinolones. These
compounds were compared with cognate N-1 cyclopropyl, C-8
methoxy fluoroquinolones for lethal activity when protein syn-
thesis was blocked by chloramphenicol. Each N-1/C-8 fused-
ring analog examined, regardless of the C-7 group or modifi-
cation to the N-1/C-8 ring, showed reduced ability to kill E. coli
in the presence of chloramphenicol. This structure-function
relationship is most easily explained by the N-1/C-8 substitu-
ents interacting with topoisomerases. However, in X-ray struc-
tures for ternary complexes (11, 26), the N-1 and C-8 positions
are far from the enzyme. We discuss the possibility that two
states exist during binding of quinolones to gyrase and topo-
isomerase IV.
acid (3B Scientific Corp., Libertyville, IL). Fused-ring derivatives were similarly
prepared using S(Ϫ)9,10-difluoro-2,3-dihydro-3-methyl-7-oxo-7H-pyrido[1,2,3-
de]-1,4-benzoxazine-6-carboxylic acid (Sigma-Aldrich, St. Louis, MO). All syn-
thetic derivatives were characterized by nuclear magnetic resonance (NMR) and
mass spectroscopy (the purity was Ն95%, as determined by analytical high-
performance liquid chromatography [HPLC]). Quinolones were dissolved in 1 N
NaOH at 1/10 final volume, followed by sterile water to give a final concentration
of 10 mg/ml. Stock solutions were kept at Ϫ20°C for several weeks during the
experiments; dilutions were prepared with sterile distilled water.
Structural models and energy minimization. Energy minimization and molec-
ular-modeling studies were performed using PC Spartan Ј06 (Wavefunction, Inc.,
Irvine, CA) and Chem3D ActiveX Pro 12.0 (CambridgeSoft). Using Spartan,
energy-minimized structures were calculated by Hartree-Fock 3-21G and
6-31G*, which are modeling methods that differ by the functions and parameters
used to calculate the equilibrium conformations and ground state energies.
Energy-minimized structures using Chem3D were calculated using MM2 param-
eters, which uses force fields parameterized primarily for conformational analysis
of small organic molecules. The dihedral angle between the quinolone core and
the C-7 piperazine ring (with rotation about the C-7–N bond) was used for
energy profile analysis. Energy profiles for different compounds were then de-
termined to identify energy maxima and minima for rotation of the C-7 rings
relative to the quinolone core. The dihedral angle for the core and piperazine
ring about the C-7–N bond was set to specified angles through 360° of rotation
(Ϫ180 to ϩ180). At each dihedral angle, the C-7–N bond was locked, energy
minimization was performed with all other atoms, and both flexible and total
energy values were calculated. This energy minimization provided lowest-energy
three-dimensional structures for each compound at each of the C-7–N bond
angles and total energy of the compound with the C-7–N bond locked at the
specified angle. The results were plotted as dihedral angle versus energy of
energy-minimized structures at each angle and fitted with a simple cubic spine
curve to provide a graphic representation of the energy maxima and minima for
the full 360° rotation of the C-7 piperazine ring. All calculations were performed
with the fluoroquinolone C-3 carboxy group in the carboxylic acid form and the
piperazine ring as the free base. The results obtained using both software pro-
grams and parameters were similar; the figures shown here were prepared using
data from the MM2-based calculations.
MATERIALS AND METHODS
Bacterial cells, culture conditions, and susceptibility testing. E. coli K-12
strain DM4100 (24) was grown on LB agar or in LB liquid medium (22). The
MIC was measured by incubation of 104 to 105 cells/ml in LB liquid medium
containing serial 2-fold dilutions of quinolone at 37°C. To measure lethal action,
cells were grown aerobically at 37°C in liquid medium to midlog phase. Solutions
of quinolone were added, and incubation was continued for 2 h. The cells were
diluted in liquid growth medium, applied to agar plates lacking the drug, and
incubated overnight at 37°C to determine the number of CFU. Percent survival
was determined relative to CFU numbers at the time of quinolone addition.
Chloramphenicol (MIC ϭ 2 ␮g/ml) was added to 20 ␮g/ml 10 min prior to the
addition of quinolone for measurement of killing in the absence of protein
synthesis.
Antimicrobial agents. Chloramphenicol and levofloxacin were obtained from
Sigma-Aldrich (St. Louis, MO); moxifloxacin, gatifloxacin, and marbofloxacin
were from Toronto Research Chemicals (Toronto, ON, Canada); rufloxacin was
from LKT Laboratories Inc. (St. Paul, MN); and pazufloxacin was from AK
Scientific Inc. (Mountain View, CA). Synthesis of compounds with different C-7

5216
MALIK ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 1. Bacteriostatic and bactericidal activities of fluoroquinolones
c
Compound
C-7 ring structure
MIC (␮g/ml)
LD99 (␮g/ml)
LD99/MIC
PD161144a
N-Ethyl piperazinyl
N-Ethyl piperazinyl
N-Methyl piperazinyl
N-Methyl piperazinyl
H-Piperazinyl
0.1
0.14
1.5
1.4
2.0
3.2
4.0
1.3
1.2
5.0
2.0
5.0
2.4
4.0
2.0
4.0
3.0
7.0
UING4-257b
UIHS-IIa-93a
Levofloxacinb
PD135042a
0.75
0.062
0.2
2.0
0.8
0.031
1.0
0.04
1.2
UING4-255b
PD161148a
H-Piperazinyl
C-Ethyl piperazinyl
C-Ethyl piperazinyl
C-Methyl piperazinyl
C-Methyl piperazinyl
Diazabicyclo
0.16
0.25
0.04
0.31
0.125
0.125
0.02
0.02
1.0
0.8
UIKRM-1-025b
Gatifloxacina
UIKRM-1-029b
Moxifloxacina
UIKRM-1-033b
Pazufloxacinb
Marbofloxacinb
Rufloxacinb
0.5
0.2
0.74
0.5
Diazabicyclo
0.25
0.08
0.06
7.0
Aminocyclopropyl
N-Methyl piperazinyl
N-Methyl piperazinyl
a Open-ring structure.
b Fused-ring structure.
c LD99, 99% lethal dose.
RESULTS
but it exhibited roughly the same bactericidal activity when
protein synthesis was blocked by chloramphenicol. In this se-
ries, no fused-ring structure was more active than an open-ring
structure in the presence of chloramphenicol (Fig. 2).
Bacteriostatic activity. The structures of compounds used in
this study are shown in Fig. 1. As shown in Table 1, the
presence of an N-1/C-8 fused ring raised the MICs by 2- to
30-fold, except when the C-7 substituent was the bicyclic ring of
moxifloxacin. In that case, no difference was observed. We
conclude that bacteriostatic activity with E. coli is generally
reduced by N-1/C-8 ring fusion. MIC values are used below to
correct lethal activity for potential differences among the com-
pounds in drug uptake, drug efflux, and formation of drug-
gyrase-DNA complexes.
Comparison of C-7 ring structures (Fig. 2) revealed the
striking effect of the N-ethyl piperazinyl group: PD161144 was
insensitive to chloramphenicol, while all other derivatives ex-
hibited reduced activity. In general, the C-7 ring structure had
a significant effect on lethal activity of the N-1-cyclopropyl-8-
methoxy derivatives when protein synthesis was blocked by
chloramphenicol. In contrast, fused-ring compounds exhibited
little difference with variation of the C-7 ring. The apparent
connection between C-7 ring structure and N-1/C-8 configura-
tion in chloramphenicol-insensitive lethality is consistent with
subsequent molecular-modeling studies (see Fig. 4 and 5), in
which steric conflict of the 1-cyclopropyl group on the 8-me-
thoxy group likely results in the 8-methoxy group impeding the
rotational freedom of the C-7 rings.
To determine whether other N-1/C-8 fused-ring fluoro-
quinolones used clinically or in veterinary medicine behave in
a manner similar to that of the levofloxacin-based derivatives,
we examined the relevant commercially available agents for
lethal action in the presence and absence of chloramphenicol.
The C-7 group of pazufloxacin, which was different from the
C-7 substituents on the other compounds, failed to impart
lethality to the levofloxacin-like fused-ring core structure in the
presence of chloramphenicol (Fig. 3). Marbofloxacin and ru-
floxacin are structural analogs of levofloxacin and UING4-257,
respectively, that have the same C-7 group but different types
of N-1/C-8 fused-ring systems (Fig. 1). These agents also
lacked substantial lethal activity with chloramphenicol-treated
cells (Fig. 3). The lethal activity of rufloxacin, the only fused-
ring derivative tested without an exocyclic methyl group, was
almost completely blocked by chloramphenicol. This result
suggested that the exocyclic methyl contributes to the lethal
action of the fused-ring structures in the absence of protein
synthesis, but it is not sufficient to achieve activity at the level
of the 8-methoxy derivative. These activity data are consistent
with modeling studies (see below) in which the exocyclic
Lethal activities of N-1 cyclopropyl, C-8 methoxy fluoro-
quinolones and cognate fused-ring derivatives. The effects of
N-1 cyclopropyl and C-8 methoxy groups on killing nongrowing
cells were first examined by comparing the lethal activities of
PD161144 and its fused-ring counterpart (UING4-257). Chlor-
amphenicol had no effect on PD161144, whereas it increased
survival with UING4-257 by 3 orders of magnitude (Fig. 2,
top). Differences among the other compound pairs were not
quite as dramatic, but they were clear. For example, UIHS-
IIa-93, which has an N-methyl piperazine at C-7, was not quite
as active in the presence of chloramphenicol as PD161144, but
it was still less affected by chloramphenicol than its N-1/C-8
fused-ring analog, levofloxacin (Fig. 2). This observation is
consistent with an early report on the effect of chlorampheni-
col on the rate of levofloxacin killing (12). PD135042, which
lacks alkyl substituents on the C-7 piperazinyl ring, required
5-times-higher concentrations to kill E. coli when chloram-
phenicol was present, but then survival dropped sharply (Fig.
2). Its fused-ring analog, UING4-255, showed little bactericidal
activity in the presence of chloramphenicol, much like that
observed with the other fused-ring compounds (Fig. 2). The
lethal activity of gatifloxacin was reduced by chloramphenicol
to about the same extent as that of the N-methyl piperazinyl
compound UIHS-IIa-93, while the fused-ring analogs exhib-
ited little lethal activity in the presence of chloramphenicol
(Fig. 2). Moxifloxacin, which has a diazabicyclo ring at C-7
instead of a piperazinyl moiety, was much more active than its
N-1/C-8 fused-ring analog in the absence of chloramphenicol,

VOL. 54, 2010
LETHALITY OF FLUOROQUINOLONES
5217
FIG. 3. Lethal activities of fused-ring fluoroquinolones that have
been used clinically. Survival of E. coli was measured as a function of
the fluoroquinolone concentration expressed as a multiple of the MIC
in the presence (filled symbols) or absence (open symbols) of chlor-
amphenicol. The names of the compounds are indicated in each panel.
Data for levofloxacin, for direct comparison, are shown in Fig. 2.
Marbofloxacin is currently used in veterinary medicine. The error bars
represent standard deviations of the means shown; similar results were
observed with 2 additional, replicate experiments.
While the N-1/C-8 ring fusion lowered fluoroquinolone-me-
diated killing when protein synthesis was blocked (Fig. 2 and
3), the lethal activity of fluoroquinolones with growing cells
was sometimes lowered, sometimes increased, and sometimes
unchanged by N-1/C-8 ring fusion, depending on the fluoro-
quinolone examined. This difference provides additional sup-
port for the existence of two lethal pathways. It also empha-
sizes that with growing cells the lethal activity of each
compound must be considered on an individual basis.
Energy minimization and modeling of C-8 methoxy quino-
lones and fused-ring analogs. To gain insight into why N-1/
C-8 fused-ring fluoroquinolones have less ability to kill in
the presence of chloramphenicol than corresponding N-1
cyclopropyl, C-8 methoxy fluoroquinolones, we compared
energy-minimized structural models of levofloxacin with
the corresponding 1-cyclopropyl-8-methoxy-fluoroquin-
olone, UIHS-IIa-93. Because levofloxacin is the single active
stereoisomer (S-isomer) of racemic ofloxacin, energy mini-
mization of 8-methoxy fluoroquinolone structures was initi-
ated with the N-1 cyclopropyl group oriented to the same
FIG. 2. Lethal activity of N-1 cyclopropyl, 8-methoxy fluoroquino-
lones and N-1/C-8 fused-ring analogs in the presence or absence of
chloramphenicol. Survival of E. coli was measured as a function of the
fluoroquinolone concentration expressed as a multiple of the MIC in
the presence (filled symbols) or absence (open symbols) of chloram-
phenicol, an inhibitor of protein synthesis. Data for the 8-methoxy
compounds are in the left column, and data for fused-ring compounds
are in the right column. The names of the compounds are indicated in
each panel (Et, ethyl; Me, methyl), as are the C-7 ring structures
(lower left). The error bars represent standard deviations of the means
shown; similar results were observed with 2 additional, replicate ex-
periments.
methyl group of the fused-ring derivatives is oriented to the
same 3-dimensional space as the N-1 cyclopropyl group in
8-methoxy fluoroquinolones, while puckering of the N-1/C-8
fused ring impinges slightly into space occupied by the 8-me-
thoxy group (Fig. 4).

5218
MALIK ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 4. Comparison of the lowest-energy structures derived from energy minimization. (A) Top view of UIHS-IIa-93 and levofloxacin showing
that the exocyclic methyl group of levofloxacin and the N-1 cyclopropyl group occupy similar 3-dimensional spaces. The 8-methoxy and N-1
cyclopropyl groups in HS-IIa-93 are rigidly trans oriented, and each is nearly perpendicular to the quinolone core. The space occupied by the
8-methoxy group of UIHS-IIa-93 is void of structure with fused-ring levofloxacin (indicated by the dashed circles). (B) Front view (carboxyl-end
view) of UIHS-IIa-93 and levofloxacin. The C-7 piperazine ring is omitted for clarity. Each structure is oriented for comparison with the 6-fluorine,
4-carbonyl, and 8-methoxy groups aligned coplanar to the quinolone core (vertical dashed lines), which reveals significant skewing of the
3-carboxylate and 1-cyclopropyl group from planarity with the core ring system for UIHS-IIa-93 due to steric repulsion of the 1-cyclopropyl and
8-methoxy groups (angled dashed line). (C) Side view of UIHS-IIa-93 and levofloxacin showing the orientation of the C-7 piperazine ring of
UIHS-IIa-93 to alleviate steric interactions with the 8-methoxy group.
face of the quinolone core as the exocyclic methyl group of
levofloxacin. Fixing the facial orientation of N-1 groups to
the active stereoisomers of chiral quinolone derivatives
maintains consistent 3-dimensional orientation of these
groups and provides the likely active rotational isomer, even
though no stereoisomers exist. As shown in Fig. 4, the 1-cy-
clopropyl and 8-methoxy groups of UIHS-IIa-93 extend
from the quinolone core ring structure in a trans configura-
tion (Fig. 4A, B, and C). The exocyclic methyl group of
levofloxacin occupies 3-dimensional space similar to that
filled by the N-1 cyclopropyl group of UIHS-IIa-93, as
shown in both front and top views (Fig. 4). A front (carbox-
yl-end) view of UIHS-IIa-93 shows that the trans-oriented
8-methoxy and 1-cyclopropyl groups possess geometries

VOL. 54, 2010
LETHALITY OF FLUOROQUINOLONES
5219
about the aryl-connected atoms to form a platform-like base
underneath the quinolone (Fig. 4B). The front view of levo-
floxacin shows that the fused ring puckers away from the
exocyclic methyl group to similarly afford a platform-like
base below the quinolone core (Fig. 4B). However, the
8-methoxy group clearly occupies 3-dimensional space that
is void of structure in the fused-ring derivatives (Fig. 4A,
dashed circles).
The fused-ring system of levofloxacin extends both N-1 and
C-8 bonds in the same plane as the quinolone core (Fig. 4).
Thus, the quinolone core ring system is nearly coplanar with
the 4-carbonyl, C-8-oxygen, 1-nitrogen, and 3-carboxylate
groups. In contrast, the conformational strain imparted by
steric clash of N-1 cyclopropyl and C-8 methoxy moieties in
UIHS-IIa-93 is relieved by forcing the trans orientation of
these groups and subsequent skewing of the quinolone core,
which breaks the coplanarity of substituents attached directly
to the quinolone core (Fig. 4B). This skewing most notably
results in the N-1 cyclopropyl bond and C-3 center deviating
ϳ20° from the C-8–O and C-6–F bonds (Fig. 4B, dashed lines).
A similar skewing of quinolone substituents was previously
shown to correlate with increased bacteriostatic activity (9). In
that situation, a highly strained conformation was induced by
steric repulsion of 8-chloro, 8-bromo, or 8-methyl groups with
an N-1-(5-amino-2,4-difluorophenyl) ring. These results dem-
onstrate that certain N-1 and C-8 groups likely affect fluoro-
quinolone activity and lethality by altering core structure (pla-
narity of the quinolone core and orientation of groups from the
core rings), in addition to binding contacts and interactions of
these groups with protein and/or DNA.
Steric effects of an N-1 cyclopropyl group that force an
8-methoxy moiety into a trans orientation were expected to
restrict movement of the methoxy group and thereby impede
free rotation of C-7 rings about the C-7–N bond. To test this
idea, we evaluated the rotational freedom of C-7 ring struc-
tures on different core structures. Two-dimensional represen-
tations of the rings and bonds rotated are shown in Fig. 5A.
After calculating the lowest-energy structures, we compared
steric energy profiles for rotation of C-7 rings about the C-7–N
bond. In this analysis, we kept the quinolone structures and
substituents rigid, except for the C-7–N bond, to approximate
rotation of the C-7 ring as if the core and its substituents were
held in place by DNA and/or protein binding contacts. As
shown for representative compounds in Fig. 5B, the energy of
rotation for the piperazine ring on ciprofloxacin, which is a
C-8–H fluoroquinolone, is uniform, with high and low energies
directly correlating to steric (gauche) interactions between the
two ring structures at 180° intervals (Fig. 5B, dotted line). In
contrast, PD161144 and UING4-257 (Fig. 5B, solid line and
dashed line, respectively) have an additional high-energy con-
formation (a high-energy barrier to rotation; peaks at Ϫ10°
and 170°) (Fig. 5B). The 8-methoxy group of PD161144 im-
parts a higher energy barrier to C-7 ring rotation than the
fused ring of UING4-257 (more than 10 kcal/mol at rotational
angles of 170° and Ϫ10° [Fig. 5B]).
FIG. 5. Calculated energies for different dihedral angles of the
quinolone core and C-7 ring about the C-7–N bond for energy-mini-
mized structures. (A) Two-dimensional representations of partial fluo-
roquinolone structures showing the three bonds (boldface) used to
calculate dihedral angles of rotation for the two ring systems about the
C-7–N bond. The structure on the left shows a dihedral angle of 0°.
Clockwise rotation of the C-7 ring to ϩ180° (curved arrow) or coun-
terclockwise rotation of the ring to Ϫ180° give an equivalent Ϯ180°
structure (right structure). (B) Plot of steric energies for 360° rotation
of the C-7 piperazine ring about the C-7–N bond from the lowest-
energy structure with no incremental energy minimization during ro-
tation for PD161144 (solid line), UING4-257 (dashed line), and cip-
rofloxacin (dotted line). (C) Plot of energies from energy-minimized
structures of PD161144 (solid line) and UING4-257 (dashed line) at
20° increments of the dihedral angle about the C-7–N bond. The three
bonds forming the dihedral angle about the C-7–N bond (shown in
panel A) were locked at 20° increments; energy minimization was then
performed to provide the lowest-energy conformation of each struc-
ture at each angle, and the total energy was calculated at each angle.
The relative energy (y axis) is the difference between the calculated
total energy at each angle and the lowest calculated energy (the angle
where the lowest-energy structure is found); the energy minimum is
thus zero for the lowest-energy structure on the relative scale.
tures at fixed dihedral angles about the C-7–N bond without
keeping the structures rigid (Fig. 5C). The energies of energy-
minimized structures of PD161144 (1-cyclopropyl-8-methoxy
structure) and UING4-257 (N-1/C-8 fused-ring structure) were
compared at 18 different dihedral angles about the C-7–N
bond between the quinolone core and the N-ethyl-piperazine
ring. Rotation about the C-7–N bond was locked (not allowed)
at each of the dihedral angles, followed by energy minimization
to obtain the lowest-energy structures and to calculate the total
molecular energy at each angle for each compound.
Once we saw that the 8-methoxy group and the N-1/C-8
fused ring impede free rotation of C-7 piperazinyl rings at
different levels, we compared the ability of each core structure
to undergo structural changes that would alleviate steric inter-
actions. For this, we performed energy minimization of struc-
As shown in Fig. 5C, the highest-energy rotational barriers
for the C-7 ring about the C-7–N bond are similar for the

5220
MALIK ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
8-methoxy and fused-ring structures (ϳ30 to 35 kcal/mol as the
dihedral angle nears Ϯ90°). In addition, the highest-energy
conformations (structures where C-7–N bond angles are at the
greatest steric conflict) in Fig. 5C are 10 to 15 kcal/mol lower
in energy than in Fig. 5B, where the structures were held rigid
at all angles. The conformational changes in structure elimi-
nated the steric conflict between the N-1/C-8 fused ring of
UING4-257 and the C-7 piperazine ring at dihedral angles of
Ϫ10° and 170° (Fig. 5B and C, dashed lines). In contrast, the
high steric conflict of the 8-methoxy group with rotation of the
C-7 ring about the C-7–N bond for PD161144 (Fig. 5B, solid
line) is still evident in the lowest-energy conformations calcu-
lated for PD161144 (Fig. 5C, solid-line peaks at 0° and 180°).
Thus, the lowest-energy rotational freedom of the C-7 piper-
azine group on the 8-methoxy fluoroquinolone is restricted to
a narrower range of angles than the C-7 piperazine group on
the fused-ring structure, which allows the C-7 group on the
fused-ring structure to more freely rotate about the C-7–N
bond over a greater range of dihedral angles (Fig. 5C). Binding
to gyrase or DNA that restricts movement of the 8-methoxy
group imparts greater restriction on the rotational freedom of
the C-7 piperazine ring of PD161144 (Fig. 5B). These results
indicate that different N-1 and C-8 groups found on different
fluoroquinolone structures likely affect activity and/or lethality
by altering the rotational and conformational properties of C-7
ring structures, in addition to direct effects of these groups on
binding to gyrase and/or DNA. We conclude that identifying
optimal C-7 rings on quinolone structures that improve killing
in the absence of protein synthesis cannot be considered inde-
pendently of core structure.
unaffected by other protein synthesis inhibitors, thereby ex-
ploring the possibility of coadministration of protein synthesis
inhibitors with quinolone-based gyrase inhibitors.
Energy minimization and modeling of the 8-methoxy fluo-
roquinolone derivatives studied above indicate that trans-ori-
ented N-1 cyclopropyl and C-8 methoxy groups form a unique
platform structure under the quinolone core that restricts the
rotational freedom of the C-7 groups and thus may combine to
more effectively destabilize the drug-gyrase-DNA complex and
result in chromosome fragmentation (2, 20). The N-1 cyclo-
propyl, C-8 methoxy fluoroquinolone PD161144 provided a
striking example of this phenomenon when rapid killing in the
presence of chloramphenicol was used as an assay (Fig. 2, top).
Changing the size or position of the alkyl group on the C-7
piperazinyl ring or fusing the C-8 methoxy and N-1 cyclopropyl
substituents enabled chloramphenicol-mediated inhibition of
protein synthesis to interfere with lethal activity. Collectively,
these data identify quinolone structure modifications that in-
fluence lethal activity in the absence of protein synthesis. En-
zyme structure is also likely to be important, since moxifloxa-
cin, rather than PD161144, is currently the compound that is
least sensitive to chloramphenicol with mycobacteria (16, 19).
Greater rotational freedom about the C-7–N bond for the
levofloxacin-like compounds than for the 8-methoxy deriva-
tives, especially when the N-1 and C-8 groups are held rigid to
approximate the quinolone core bound to protein and/or
DNA, suggests that the rotational and conformation freedom
of C-7 groups is an important factor in explaining why different
C-7 groups significantly affected chloramphenicol-insensitive
killing by N-1 cyclopropyl, 8-methoxy quinolones while little
effect was observed with the fused-ring derivatives (Fig. 2). We
hypothesize that the unique ability of N-1 cyclopropyl, C-8
methoxy fluoroquinolones substituted with select C-7 struc-
tures to kill E. coli in the absence of protein synthesis is due to
multiple structural factors that include (i) overall changes in
the molecular geometry of the quinolone core and substituents
due to C-8 and N-1 groups, (ii) the trans-oriented platform
under the quinolone core interacting with gyrase, and (iii)
impeded or altered rotational and conformational properties
of the C-7 group affording a more rigid drug-gyrase-DNA
binding interaction.
X-ray structures have been reported for fluoroquinolone-
topoisomerase IV-DNA complexes (11, 26), but they fail to
provide a simple explanation for the lethal effects of selected
combinations of N-1 and C-8 groups. Since two binding states
are observed with kinetic experiments (8), it is likely that the
observations in the present work apply to a binding state not
yet revealed by X-ray structure analysis. To explain why rapid
killing by quinolones requires higher concentrations than are
needed to block growth (for example, Fig. 3), we speculate that
both binding sites must be filled for killing while only one must
be filled to block growth. Ongoing studies include (i) further
covariation of N-1 and C-8 structures of various quinolone
core structures to more fully understand the structural require-
ments at these positions to kill nonreplicating cells and (ii)
direct tests of the hypothesis that destabilization of DNA-
gyrase complexes by fluoroquinolones accounts for the death
of nongrowing cells.
DISCUSSION
Rapid killing of bacterial pathogens by fluoroquinolones is
thought to be important in patient treatment (1) and in re-
stricting the emergence of resistance (15, 17). Killing nongrow-
ing bacteria is likely to be particularly important with patho-
gens that form biofilms and/or enter dormant states. The
observation that quinolones kill by two pathways and that the
drug structure influences the relative use of the pathways pro-
vides an opportunity to study the pathways by examining the
effects of quinolone structure. The pathway occurring in the
absence of oxygen or protein synthesis, which is restricted to a
subset of fluoroquinolones, may arise from destabilization of
quinolone-gyrase-DNA complexes. Since DNA in the com-
plexes is broken, destabilization could lead to chromosome
fragmentation and cell death (2, 20). The present work used
rapid killing of E. coli in the presence of chloramphenicol as a
physiological readout to examine the effect of the quinolone
structure at positions N-1, C-8, and C-7 on possible destabili-
zation of gyrase-DNA complexes. The ability of quinolones to
kill E. coli in the absence of protein synthesis was impaired by
fusion of a C-8 oxygen and N-1 alkyl into a constrained ring
system (Fig. 2 and 3). Experiments with other bacteria are now
required to determine whether the widely used compound
levofloxacin is suboptimal with bacterial pathogens, such as M.
tuberculosis, that are likely to enter a dormant state. Additional
experiments will also reveal whether killing by PD161144 is

VOL. 54, 2010
LETHALITY OF FLUOROQUINOLONES
5221
ACKNOWLEDGMENTS
enoxacin and lomefloxacin against Escherichia coli KL16. Eur. J. Clin. Mi-
crobiol. Infect. Dis. 8:731–733.
15. Louie, A., H. Heine, K. Kim, D. Brown, B. VanScoy, W. Liu, M. Kinzig-
Schippers, F. So¨rgel, and G. Drusano. 2008. Use of an in vitro pharmaco-
dynamic model to derive a linezolid regimen that optimizes bacterial kill and
prevents emergence of resistance in Bacillus anthracis. Antimicrob. Agents
Chemother. 52:2486–2496.
We thank Arkady Mustaev and Xilin Zhao for critical comments on
the manuscript.
The work was supported by NIH grants R01-AI73491 and R01-
AI87671.
16. Malik, M., and K. Drlica. 2006. Moxifloxacin lethality with Mycobacterium
tuberculosis in the presence and absence of chloramphenicol. Antimicrob.
Agents Chemother. 50:2842–2844.
17. Malik, M., G. Hoatam, K. Chavda, R. Kerns, and K. Drlica. 2010. Novel
approach for comparing quinolones for emergence of resistant mutants
during quinolone exposure. Antimicrob. Agents Chemother. 54:149–156.
18. Malik, M., S. Hussain, and K. Drlica. 2007. Effect of anaerobic growth on
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19. Malik, M., T. Lu, X. Zhao, A. Singh, C. Hattan, J. Domagala, R. Kerns, and
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20. Malik, M., X. Zhao, and K. Drlica. 2006. Lethal fragmentation of bacterial
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