Design, synthesis, and evaluation of novel fluoroquinolone - Aminoglycoside hybrid antibiotics

Article abstract of DOI:10.1021/jm900028n

A series of new hybrid structures containing fluoroquinolone (ciprofloxacin) and aminoglycoside (neomycin) antibiotics linked via 1,2,3-triazole moiety were designed and synthesized, and their antibacterial activities were determined against both Gram-neg

Full text of DOI:10.1021/jm900028n

J. Med. Chem. 2009, 52, 2243–2254
2243
Design, Synthesis, and Evaluation of Novel Fluoroquinolone-Aminoglycoside Hybrid
Antibiotics
Varvara Pokrovskaya,† Valery Belakhov,† Mariana Hainrichson,† Sima Yaron,‡ and Timor Baasov*,†
The Edith and Joseph Fisher Enzyme Inhibitors Laboratory, Schulich Faculty of Chemistry, and Department of Biotechnology and Food
Engineering, TechnionsIsrael Institute of Technology, Haifa 32000, Israel
ReceiVed January 12, 2009
A series of new hybrid structures containing fluoroquinolone (ciprofloxacin) and aminoglycoside (neomycin)
antibiotics linked via 1,2,3-triazole moiety were designed and synthesized, and their antibacterial activities
were determined against both Gram-negative and Gram-positive bacteria, including resistant strains. The
nature of spacers in both the ciprofloxacin and neomycin parts greatly influenced the antibacterial activity.
The majority of hybrids was significantly more potent than the parent neomycin and overcame most prevalent
types of resistance associated with aminoglycosides. Selected hybrids inhibited bacterial protein synthesis
with the potencies similar to or better than that of neomycin and were up to 32-fold more potent inhibitors
than ciprofloxacin for the fluoroquinolone targets, DNA gyrase and toposiomerase IV, indicating a balanced
dual mode of action. Significant delay of resistance formation was observed in both E. coli and B. subtilis
to the treatment with ciprofloxacin-neomycin hybrid in comparison to that of each drug separately or their
1:1 mixture.
Introduction
in Enterococcus,4 Staphylococcus,5 and Streptococcus3 isolates,
including the methicillin-resistant Staphylococcus aureus
(MRSA),6 and has been the most extensively investigated
because of the large number of clinically important aminogly-
cosides that are susceptible to modification with this enzyme.7,8
The increasing emergence of antibiotic resistance in patho-
genic bacteria to current antibiotics is of growing importance
worldwide. Among the different classes of clinically important
antibiotics that largely suffered from the resistance problem
during the past few decades is the aminoglycoside class of drugs.
These antibiotics have broad-spectrum of activity against both
Gram-negative and Gram-positive bacteria by selectively target-
ing bacterial protein synthesis machinery and have been used
for over 50 years. Such a prolonged clinical and veterinary use
of currently available aminoglycosides has resulted in effective
selection of resistance, which severely limits their usefulness.1
The most prevalent mechanism in clinical isolates of resistant
bacteria is the bacterial acquisition of aminoglycoside-modifying
enzymes, which modify the antibiotics by N-acetyltransferase
(AACa), O-phosphotransferase (APH), and O-nucleotidylyl-
transferase (ANT) activities.2 Among these enzymes families,
aminoglycoside 3′-phosphotransferases [APH(3′)’s], of which
seven isozymes are known, are widely represented. These
enzymes catalyze phosphorylation at the 3′-OH of both neo-
mycin and kanamycin classes of aminoglycosides, rendering the
resulting phosphorylated products inactive.
To tackle the problem of bacterial resistance caused by
enzymatic modification, many analogues of aminoglycosides
have been synthesized by direct chemical modification of
existing aminoglycoside drugs.9,10 Earlier investigations in this
direction have yielded several semisynthetic drugs such as
amikacin, dibekacin, and arbekacin.9,11 However, new resistance
to these drugs has emerged soon after their introduction to the
clinic.1,12 Therefore, there is an urgent need for new classes of
aminoglycosides that are active against known and spreading
resistance mechanisms. One such strategy that has been pursued
in recent years employs a combination of two different drugs
in one molecule.13 With this strategy, each drug moiety is
designed to bind independently to two different biological targets
and synchronously accumulate at both target sites. Such dual
action drugs, or hybrid drugs, offer the possibility to overcome
the current resistance and in addition to reduce the appearance
of new resistant strains.14 Several successful applications of
hybrid drugs approach have been reported.15-17 The dual action
compounds, combining fluoroquinolone (enrofloxacin or nor-
floxacin) and cephalosporin (cefamandole) moieties with an
amide linkage, were particularly potent against Enterobacter
species.16 Fluoroquinolone-anilinouracil hybrids linked via their
secondary amino groups have also been synthesized.17 A series
of oxazolidinone-quinolone hybrid structures, which simulta-
neously act on two different cellular functions, DNA replication
and protein synthesis, have been reported.14,18 The best com-
pounds of this series exhibited a balanced dual mode of action
and overcame the majority of known resistance mechanisms to
quinolones and linezolid in clinically relevant Gram-positive
pathogens.
Although most of these enzymes are typically monofunctional
enzymes, the recent emergence of genes encoding bifunctional
aminoglycoside-modifying enzymes is another sophistication
relevant to the clinical use of aminoglycosides.3 Among them,
the bifunctional AAC(6′)/APH(2′′) enzyme has been detected
* To whom correspondence should be addressed. Phone: (+972) 4829-
2590. Fax: (+972) 4829-5703. E-mail: chtimor@tx.technion.ac.il.
†
The Edith and Joseph Fisher Enzyme Inhibitors Laboratory.
Department of Biotechnology and Food Engineering.
‡
a Abbreviations: AAC, aminoglycoside N-acetyltransferase; APH, ami-
noglycoside O-phosphotransferase; ANT, aminoglycoside O-nucleotidy-
lyltransferase; MRSA, methicillin-resistant Staphylococcus aureus; NeoB,
neomycin B; Cipro, ciprofloxacin; TIPSCl, triisopropylchlorosilane; PMB,
p-methoxybenzyl; TBAF, tetra-n-butylammonium fluoride; CAN, cerium
ammonium nitrate; DCC, N,N′-dicyclohexylcarbodiimide; TEMPO, 2,2,6,6-
tetramethylpiperidine-1-oxyl; BAIB, [bis(acetoxy)iodo]benzene; HOBT,
hydroxybenzotriazole; MIC, minimal inhibitory concentration; TopoIV,
topoisomerase IV; IC50, half-maximal inhibitory concentration.
We previously reported that the modification of clinical
aminoglycoside neomycin B (NeoB) by linking a variety of
sugars at C5′′-OH group via glycosidic linkage resulted in a
10.1021/jm900028n CCC: $40.75
2009 American Chemical Society
Published on Web 03/20/2009

2244 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
PokroVskaya et al.
The present work describes the synthesis and biological
evaluation of the Cipro-NeoB hybrids (1), where the two
pharmakophores are linked through different spacers: spacer X
in the Cipro part and spacer Y in the NeoB part. We report that
selected hybrid structures have high potency against both Gram-
negative and Gram-positive bacteria including MRSA and
strains harboring either the monofunctional APH(3′) enzyme
or the bifunctional AAC(6′)/APH(2′′) enzyme. We also show
that new hybrids exhibit the dual mode of action by inhibiting
both targets: bacterial protein synthesis and topoisomerase/
gyrase.
Chemistry
The synthesis of the Cipro-NeoB hybrids 1 is described in
Scheme 1. We separately prepared nine azido derivatives of
Cipro (compounds 2a-i) and three different alkyne derivatives
of NeoB (compounds 3a-c), which were then coupled via “click
reaction”25 to afford a library of 17 different Cipro-NeoB
hybrids 1a-q. The spacers X and Y were selected to vary both
the length and chemical nature of the linkage between the two
pharmakophores Cipro and NeoB.
Several SAR studies on fluoroquinolones have demonstrated
a high tolerance for structural variations at the 7-position of
the phenyl ring (Figure 1), including alkylations at the terminal
nitrogen of the piperazine moiety.14,16-18,26,27 On the basis of
this information, we choose to modify Cipro at the terminal
nitrogen of the piperazine moiety with various linkers containing
an azide group. The derivatives 2a-i were prepared by direct
coupling of the commercial Cipro with the corresponding
bromoazides/chloroazides under reflux and base conditions
(NaHCO3, CH3CN), and the yields of these products are
summarized in Table 1. The bromoazides/chloroazides were
synthesized from the corresponding dibromo or bromochloro
compounds according to published procedure.28
The synthesis of the alkyne derivatives of NeoB (compounds
3a-c) is described in Scheme 2. For the preparation of the
alkyne derivative 3a, commercial NeoB was first converted to
the corresponding per-azido derivative according to published
procedure,29 followed by selective protection of the primary
hydroxyl to afford the intermediate 4. Protection of all the
secondary hydroxyls with p-methoxybenzyl (PMB) ether and
selective removal of silyl ether with TBAF gave compound 5.
Treatment of 5 with propargyl bromide under base conditions
of NaH afforded the 5′′-alkyne derivative 6. Two deprotection
steps, removal of PMB ether protections with CAN followed
by Staudinger reaction to convert all the azides to the corre-
sponding amines, gave the alkyne derivative 3a.
Figure 1. Structures of ciprofloxacin, neomycin B, and the designed
hybrids 1.
new class of pseudopentasaccharides that exhibited similar or
better antibacterial activities to that of the parent NeoB against
selected bacterial strains.19 However, while the specificity
constant values (kcat/Km) of these derivatives with the aminogly-
coside resistance enzyme APH(3′)-IIIa were in general lower
than that of NeoB, the best compounds exhibited these values
only about 10-fold lower than that of NeoB, suggesting that
several different conformations of the designed structures can
bind productively the APH(3′)-IIIa and lead to the enzyme-
catalyzed phosphoryl transfer process. Similar superb substrate
promiscuity of APH(3′)-IIIa with other nonsugar modifications
of NeoB at the C5′′ position has been recently reported,20
although several of these derivatives exhibited enhanced anti-
bacterial activity compared to the parent NeoB. To further
explore the modification of NeoB at the C5′′ position to
maximize the ability of new derivatives to compromise with
the activity of APH(3′) and other aminoglycoside-modifying
enzymes, we undertook additional synthesis of this class of
compounds. Specifically, having been encouraged by the
recently reported potential of hybrid antibiotics, we have
prepared a new hybrid structures of NeoB and the fluoroqui-
nolone-ciprofloxacin (Cipro) linked via 1,2,3-triazole moiety,
which we called Cipro-NeoB hybrids (compounds 1, Figure
1).
Quinolones exert their antibacterial activity by targeting
bacterial DNA gyrase and topoisomerase IV (TopoIV) and
inhibiting DNA replication process.21 Specifically, they bind
to complexes that form between DNA and DNA gyrase or
TopoIV. The quinolone-gyrase-DNA or quinolone-TopoIV-
DNA complex formation inhibits DNA replication and cell
growth and is responsible for the bactericidal action of quino-
lones.22 On the basis of these data, we hypothesized that the
Cipro-NeoB hybrids (1), because of the presence of the highly
positively charged NeoB, could afford favorable binding to that
of Cipro to these ternary complexes by forming additional
contacts to DNA and/or DNA-protein interface and as such
exhibit better inhibition and improved antibacterial activity. The
well-established binding of aminoglycosides to DNA,23 along
with the inhibition of various nucleic acid metabolizing enzymes
by aminoglycosides,24 supported this hypothesis.
Two other alkyne derivatives of NeoB, compounds 3b,c,
contain a terminal alkyne group connected to the NeoB moiety
at 5′′-position via an amide linkage (Scheme 2b). For the
assembly of these derivatives, the readily available 5′′-alcohol
7
30 was converted to the corresponding 5′′-acid 8 according to
the published procedure.31 The resulting acid was then coupled
with the commercially available alkyne-amines in the presence
of DCC to afford the corresponding amides 9a,b in reasonable
yields. Finally, removal of all the ester protections (MeNH2,
MeOH) and Staudinger reaction yielded the alkyne derivatives
3b,c in excellent isolated yields.
The key coupling reaction between NeoB-alkyne derivatives
(compounds 3a-c) and Cipro-azide derivatives (compounds
2a-i) was performed under microwave irradiation (∼40 s) in
the presence of organic base (7% Et3N in water) and the Cu(I)
catalyst to ensure the production of a single (anti) stereoisomer
at the triazole moiety.25,32 The reaction proceeded almost

Fluoroquinolone-Aminoglycoside Hybrid Antibiotics
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2245
Scheme 1. Strategy for the Assembly of Cipro-NeoB Hybrids 1a-qa
a (a) [(CH3CN)4Cu]PF6, 7% Et3N in water, microwave irradiation 40 s.
Table 1. Chemical Yields and Preparation of Ciprofloxacin-Azido
Derivatives 2a-i
causes of bacterial infections and exerts high level of resistance
to aminoglycosides.34
As can be deduced from the MIC data, all of the synthesized
hybrids exhibited significant antibacterial activity. This activity
was especially improved in comparison to that of NeoB, while
the activity of all the hybrids was lower than that of Cipro. The
most prominent improvement was observed against all E. coli
strains including aminoglycosides-resistant strains E. coli AG100A
and E. coli AG100B. On average, the hybrids showed 2-8 and
2-16 times better activity than NeoB against E. coli R477-100
and E. coli 25922, respectively, and this was much higher
against the resistant E. coli AG100A and E. coli AG100B strains
with 1i and 1q as the most active derivatives. Compound 1i
was 128-fold more potent than NeoB against E. coli AG100B
and E. coli AG100A; compound 1q was 32-fold better than
NeoB against E. coli AG100B and 253-fold better against E.
coli AG100A. Unlike the activity against Gram-negative E. coli,
most of the hybrids were less active than the parent NeoB
against the Gram-positive Bacillus subtilis, susceptible to
aminoglycosides. However, all the hybrids displayed signifi-
cantly better potency against the aminoglycosides-resistant
Gram-positive MRSA with the activities of 8- to 128-fold better
than that of NeoB. The hybrid compounds 1i and 1q retained
activity similar to that of NeoB in B. subtilis and displayed the
most prominent activity against the MRSA.
azido linker coupled
compd
with Ciproa
X
yield (%)
2a
2b
2c
2d
2e
2f
2g
2h
2i
(Cl)Br-(CH2)2-N3
(Cl)Br-(CH2)3-N3
(Cl)Br-(CH2)4-N3
(Cl)Br-(CH2)5-N3
-(CH2)2-
-(CH2)3-
-(CH2)4-
-(CH2)5-
-(CH2)6-
75
70
80
65
57
31
75
44
30
Br-(CH2)6-N3
Br-CH2CH(OH)CH2-N3
Br-(CH2)2-O-(CH2)2-N3
Br-CH2-mC6H4-CH2-N3
Br-CH2-pC6H4-CH2-N3
-CH2CH(OH)CH2-
-(CH2)2-O-(CH2)2-
-CH2-mC6H4-CH2-
-CH2-pC6H4-CH2-
a The azido linkers were prepared from the corresponding commercially
available dibromo or bromochloro compounds according to a published
procedure.28
quantitatively in the presence of 1.2 equiv of Cipro-azide
derivative and 1 equiv of NeoB-alkyne. The unreacted Cipro-
azide could then be easily separated from the hybrid product
by passing the reaction mixture through a short column of
Amberlite CG-50 (H+ form) resin. As test cases indicated that
the protocol provided excellent yields of highly pure product,
the three NeoB-alkyne derivatives 3a-c were coupled with
selected Cipro-azide derivatives 2a-i to produce 17 hybrid
products 1a-q (Table 2); all compounds were synthesized at a
minimum of a 0.05 mmol scale to provide approximately 50
mg of each product (as a free base form). The structures of
1a-q were confirmed by a combination of various 1D and 2D
As to the SAR among the 17 hybrid structures, it appears
that the length of the linear aliphatic chain at position X (1a-e
and 1j-l) has no particular influence on antibacterial activity,
as the variation in antibacterial potency against individual strains
is very little. This is valid when comparing between 1a-e or
between 1j-l of the same sets with respect to the Y spacer or
between 1a-e and 1j-l of different sets. Hybrids consisting
of the linear aliphatic chain incorporating alcohol functionality
(1f and 1m) show better activity than those incorporating ether
functionality (1g and 1n) at position X. Among the derivatives
containing an aromatic linker at position X (1h,i and 1o,p), the
derivative 1i that contains para-substituted benzene ring at both
X and Y positions displayed the most prominent activity against
all bacterial strains tested. Surprisingly, the only hybrid that
displayed a spectrum of activity similar to that of 1i was the
hybrid 1q that contains the shortest linkers at both X and Y
positions. Nevertheless, the observation that the majority of
hybrids are more active than NeoB against Gram-negative
bacteria (E. coli) while retaining moderate activities against the
susceptible Gram-positive bacteria (B. subtilis) prompted us to
further investigate the activity of hybrids against E. coli strains
harboring particular aminoglycosides resistant plasmids.
NMR techniques, including 2D H-13C HMQC and HMBC,
2D COSY, and 1D selective TOCSY experiments, along with
mass spectral analysis.
1
Results and Discussion
The Cipro-NeoB hybrids 1a-q were tested for in vitro
antibacterial activity against a panel of susceptible and resistant
bacterial strains using Cipro and NeoB as controls (Table 2).
Data for selected Gram-negative and Gram-positive strains are
reported as minimal inhibitory concentration (MIC) values.
Resistant strains included Escherichia coli AG100A and AG100B
(Gram-negative) and methicillin-resistant S. aureus (MRSA)
(ATCC 43300, Gram-positive). E. coli AG100A and AG100B
are aminoglycosides-resistant laboratory strains that harbor Kanr
transposon Tn903.33 MRSA (ATCC 43300) is one of the leading
For this purpose, we tested selected hybrids against five
isogenic E. coli strains (Table 3). E. coli (pSF815) and E. coli
(pET9d) are laboratory resistant strains derived by transforma-

2246 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
Scheme 2. Synthesis of NeoB 5′′-Alkyne Derivatives
PokroVskaya et al.
tion of E. coli XL1 blue (background strain) with the pSF815
and pET9d plasmids, respectively. The pSF815 encodes for the
bifunctional AAC(6′)/APH(2′′) resistance enzyme, which cata-
lyzes acetylation of the amino group at 6′-NH2 and phospho-
rylation at the 2′′-OH. The pET9d encodes for the APH(3′)-Ia
resistance enzyme, which catalyzes phosphorylation at the 3′-
OH of both neomycin and kanamycin families of aminoglyco-
sides. The last two isogenic strains used were E. coli BL21
(background strain) and E. coli (pETSACG1). The latter was
derived by transformation of E. coli BL21 with the pETSACG1
plasmid that encodes for the APH(3′)-IIIa resistance enzyme.
These three enzymes are among the most prevalent modes of
resistance found in aminoglycosides resistance strains.5,34,35,2
Since the aminoglycoside resistance of the engineered strains,
E. coli (pSF815), E. coli (pET9d), and E. coli (pETSACG1), is
mediated only because of the presence of the respective cloned
resistance enzyme, comparison of the MIC values against each
pair of the resistant and background strains eliminates other
effects that could affect the activity of the tested compound,
like penetration or solubility. Consequently, a poorer substrate
for the resistance enzyme should have a low ratio between the
MIC values of the resistant and nonresistant strains, as
demonstrated in several earlier studies.19,20,36
The data in Table 3 illustrate that the MIC values for NeoB
are >64-, 16-, and 8-fold higher for the resistant strains than
the respective nonresistant strains. As expected, the activity of
NeoB against the resistant strain harboring bifunctional AAC(6′)/
APH(2′′) resistance is significantly lower than those harboring
monofunctional APH(3′)-Ia or APH(3′)-IIIa resistance. All the
tested hybrids were less effective than Cipro but displayed
significant to excellent activities against resistant strains with
the potencies far greater than that of the parent NeoB. The hybrid
structures also displayed similar (E. coli XL1 blue) or better
(E. coli BL21) antibacterial efficiency compared to NeoB against
susceptible strains. Subsequently, the MIC ratio for each tested
hybrid (calculated by dividing the MIC value against resistant

Fluoroquinolone-Aminoglycoside Hybrid Antibiotics
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2247
Table 2. Chemical Yields and MICs of the Hybrids 1a-q against Selected Bacterial Strainsa
MIC (µg/mL)b
E.coli
R477-100
E.coli
ATCC 25922
E.colic
AG100B
E.colic
AG100A
B. subtilis
ATCC 6633
MRSA ATCC
43300
compd
X
Y
yield (%)
Cipro
NeoB
1a
1b
1c
1d
1e
1f
1g
1h
1i
1j
1k
1l
0.02
24
12
6
12
12
6
6
24
6
1.5
12
12
24
6
12
6
6
0.02
48
6
3
6
6
6
3
12
12
3
6
6
6
3
6
6
0.05
384
24
12
24
24
12
6
<0.005
96
0.75
1.5
1.5
1.5
1.5
0.75
1.5
1.5
0.75
1.5
1.5
1.5
0.75
1.5
0.75
1.5
0.02
1.5
6
6
6
12
12
6
12
6
1.5
3
6
12
3
12
3
3
0.75
0.20
384
48
48
24
48
48
24
24
12
3
24
48
48
12
24
6
-(CH2)2-
-(CH2)3-
-(CH2)4-
-(CH2)5-
-(CH2)6-
-CH2CH(OH)CH2-
-(CH2)2-O-(CH2)2-
-CH2-mC6H4-CH2-
-CH2-pC6H4-CH2-
-(CH2)2-
-C6H4-NHCO-
-C6H4-NHCO-
-C6H4-NHCO-
-C6H4-NHCO-
-C6H4-NHCO-
-C6H4-NHCO-
-C6H4-NHCO-
-C6H4-NHCO-
-C6H4-NHCO-
-CH2-NHCO-
-CH2-NHCO-
-CH2-NHCO-
-CH2-NHCO-
-CH2-NHCO-
-CH2-NHCO-
-CH2-NHCO-
-CH2-O-CH2-
63
90
92
94
96
92
95
95
91
97
93
96
85
90
93
96
97
12
12
3
12
12
24
12
12
12
12
12
-(CH2)3-
-(CH2)5-
1m
1n
1o
1p
1q
-CH2CH(OH)CH2-
-(CH2)2-O-(CH2)2-
-CH2-mC6H4-CH2-
-CH2-pC6H4-CH2-
-(CH2)2-
6
3
3
6
3
0.38
a The boldface rows in the table highlight the most potent compounds. b The MIC values represent the results obtained in parallel experiments with two
different starting concentrations of the tested compound (384 and 1.5 µg/mL). c Kanamycin resistant Escherichia coli strains expressing APH(3′)-I aminoglycoside
resistance enzyme.33
Table 3. Antibacterial Activities of Selected Hybrids against E. coli XL1 Blue and E. coli BL21 (Background Strains) and Their Engineered Variants
MIC (µg/mL)
MIC (µg/mL)
MIC (µg/mL)
XL1 blue/pSF815, expressed MIC
enzyme AAC(6′)-APH(2′′)
XL1 blue/pET9d,
MIC
BL21/pETSACG1, expressed MIC
compd XL1 blue
ratioa expressed enzyme APH(3′)-Ia ratioa
BL21
enzyme APH(3′)-IIIa
ratioa
Cipro
NeoB
1i
1m
1o
1p
1q
0.10
6
3
6
6
0.38
>384
24
48
24
3.8
>64
0.10
96
3
12
6
1
16
1
2
1
<0.005
6
<0.005
48
0.4
0.2
0.4
1
8
1
1
1
1
1
8
8
4
1
4
0.4
0.2
0.4
0.4
0.2
6
3
6
12
6
12
1
4
0.4
0.2
a The MIC ratios were calculated by dividing the MIC value against resistant strain by that against the respective background strain.
strain to the MIC value against susceptible strain) was signifi-
cantly lower than that calculated for the NeoB. Most importantly,
this MIC ratio was 1 for the majority of cases: for all the hybrids
against E. coli (pETSACG1); for the hybrids 1i, 1p, and 1o
against E. coli (pET9d); and for the 1p against E. coli (pSF815).
The observed identical MICs of the hybrids against different
isogenic pairs of bacteria indicate that the reason for the
observed sensitivity of the E. coli harboring AAC(6′)/APH(2′′)
(in the case of 1p), APH(3′)-Ia (in the cases of 1i, 1p, and 1o),
or APH(3′)-IIIa (in all the tested hybrids) is the inferior activity
of these enzymes toward particular hybrids rather than reduced
permeability of the hybrid structures.
To further substantiate this observation, a detailed kinetic
analysis of the purified APH(3′)-IIIa enzyme with NeoB along
with the selected hybrid 1m (that displayed the MIC ratio of 1
against pETSACG1) was carried out according to the previously
reported procedure.19 The measured Km (µM), kcat (s-1), and
kcat/Km (M-1 ·s-1) values were 5.7 ( 0.7, 2.4 ( 0.1, and 42 ×
10-4 for NeoB and 86.8 ( 8.9, 2.5 ( 0.1, and 2.9 × 10-4 for
the hybrid 1m. The kinetic constants measured for NeoB are
similar to previously reported values.19,37 The observed data
indicate that the hybrid 1m is a poorer substrate of APH(3′)-
IIIa than NeoB. The observed decrease in specificity for 1m is
caused primarily by its poor ability to saturate the enzyme, as
judged from its elevated Km (87 µM) compared to that of the
NeoB (Km ) 6 µM). The observed kinetic data with 1m are
consistent with the antibacterial data (Table 3). In fact,
comparison of its Km value (87 µM) with the MIC of 5.5 µM
(0.2 µg/mL) against E. coli (pETSACG1) suggests that the
bacteria are killed at far lower concentration before the enzyme’s
full activity is reached.
From the data in Table 3 it was interesting to find out that
the MIC ratio for Cipro in the case of the isogenic E. coli XL1
blue (pSF815)/E. coli XL1 blue strains was 3.8, indicating a
modification of this important clinical antibiotic by the ami-
noglycoside resistant AAC(6′)/APH(2′′) enzyme. Since two

2248 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
PokroVskaya et al.
Table 4. Activity of Selected Hybrids as Inhibitors of DNA Gyrase,
TopoIV, and Bacterial Protein Synthesis
Nevertheless, the observed superior activity of the selected
hybrids (like 1i and 1q) compared to that of the parent NeoB
against a variety of Gram-negative and Gram-positive bacteria
including resistant strains along with their established dual mode
of action warrants further investigation of these compounds.
Finally, one advantage of hybrid drugs is their potential to
slow the emergence of resistance.13,17 The underlying hypothesis
is that treatments that inhibit multiple targets might delay and
decrease the pathogen’s ability to accumulate simultaneous
mutations that affect the multiple targets.41 To assess the
potential of emergence of antibacterial resistance to Cipro-NeoB
hybrids, we used a procedure of selective pressure in which
both E. coli ATCC 35218 and B. subtilis ATCC 6633 were
exposed to subinhibitory (1/2 MIC) concentrations of Cipro,
NeoB, Cipro/NeoB mixture (1:1 molar ratio), and hybrid 1i
during 15 successive subcultures (Figure 3). We note that the
MIC values of the Cipro/NeoB mixture (1:1 molar ratio) against
the bacterial strains mentioned in Table 2 were very similar to
that of Cipro (on the weight basis of the composition of Cipro
in the mixture; data not shown). Therefore, in these experiments
we were very interested, in addition to Cipro and NeoB, to also
include the mixture Cipro + NeoB. As can be seen from the
data in Figure 3, the relative MIC values of Cipro, NeoB, and
Cipro + NeoB mixture increased by 75-, 4-, and 20-fold against
E. coli and by 37.5-, 8-, and 7.6-fold against B. subtilis while
that of the hybrid 1i remained unchanged against both E. coli
and B. subtilis. Similar emergence of resistance under same
experimental conditions has been reported for Cipro42 and
aminoglycosides.43 To our knowledge, the ability of hybrid
drugs to delay the emergence of resistance development was
notdemonstratedforthepreviouslyreportedhybridstructures.13-18
As such, the observed delay of resistance development to the
hybrid 1i, compared to that of Cipro, NeoB, and the mixture
Cipro + NeoB in both the Gram-negative (E. coli) and Gram-
positive (B. subtilis) bacteria, is very encouraging.
IC50 (µM)
compd
DNA gyrasea
TopoIVb
protein synthesisc
Cipro
NeoB
1f
1i
1q
1.3 ( 0.1
10.8 ( 0.3
inactive
0.58 ( 0.04
0.55 ( 0.06
7.90 ( 0.25
inactive
inactive
10.5 ( 0.1
2.2 ( 0.6
16.7 ( 4.4
18.1 ( 4.9
0.073 ( 0.005
0.085 ( 0.003
0.041 ( 0.009
a Supercoiling assay with E. coli DNA gyrase. The IC50 was defined as
the drug concentration that reduced the enzymatic activity observed with
drug-free controls by 50%. See Experimental Section for assay details.
b Relaxation assay with E. coli TopoIV. The IC50 was defined as the drug
concentration that reduced the enzymatic activity observed with drug-free
controls by 50%. See Experimental Section for assay details. c In vitro
transcription/translation assay with E. coli S30 extract system. See
Experimental Section for assay details.
other strains harboring APH(3′) activity displayed MIC ratios
of 1, we anticipated that Cipro may undergo N-acetylation at
the terminal nitrogen of its piperazine moiety by AAC(6′)/
APH(2′′). This suggestion is supported by recent reports
demonstrating that some common aminoglycoside acetyltrans-
ferases (AACs), including AAC(6′)s, are capable of performing
N-acetylation of flouroquinolones having a free amino group
at 7-position.38,39 In addition, a close inspection of the data in
Table 2 reveals that Cipro displayed particularly reduced activity
against S. aureus (MRSA): MIC of 0.2 in comparison to MIC
values of 0.05 to <0.005 against other tested strains. Since the
bifunctional AAC(6′)/APH(2′′) enzyme is the most frequently
encountered aminoglycoside-modifying enzyme in staphylo-
cocci, including MRSA,6 the observed reduced activity of Cipro
against S. aureus (MRSA) may be due to the modification of
Cipro by AAC(6′)-APH(2′′). To further investigate these
observations, purification of the bifunctional AAC(6′)/APH(2′′)
and the detailed kinetic analysis of Cipro with the homogeneous
enzyme, along with the structural characterization of the
enzymatic product, are currently underway.
Conclusions
To investigate the possibility of a dual mode of action, we
measured for the hybrids 1f, 1i, and 1q both the inhibition of
protein synthesis in an in vitro transcription/translation assay
and the inhibition of the enzymes that are targeted by the
quinolones, DNA gyrase, and TopoIV (Table 4 and Figure 2).
The observed data show that the hybrid compounds inhibited
bacterial protein synthesis with potencies similar to or better
than that of NeoB, confirming their strong aminoglycoside
character and the observed antibacterial activity. On the basis
of the observed reduced antibacterial activity of all the hybrids
in comparison to that of Cipro (Tables 2 and 3), we expected
that the hybrids should be weaker DNA gyrase and TopoIV
inhibitors than Cipro. However, the hybrids 1f, 1i, and 1q
displayed far greater activities than Cipro in both the DNA
gyrase and TopoIV assays, indicating the dual mode of action
of these molecules. The measured IC50 values for 1f, 1i, and
1q were 18-, 15-, and 32-fold superior to that of Cipro in DNA
gyrase assay and 19-, 20-, and 1.4-fold superior to Cipro in
TopoIV assay. It is of note that the IC50 values determined for
Cipro for the inhibition of DNA gyrase and TopoIV are very
similar to those previously reported.18,40 These data clearly
confirm our design principle of the Cipro-NeoB hybrids and
their desired dual mode of action. The observed difference
between antibacterial performance and targets inhibition can be
best explained by the reduced cell penetration of the hybrid
structures in comparison to Cipro. Both the higher molecular
weight and the higher charge of the hybrids compared with those
of Cipro might contribute to their reduced cell penetration.
A series of hybrid compounds containing a covalently linked
fluoroquinolone (Cipro) and aminoglycoside (NeoB) with potent
antibacterial activity and dual mode of action has been
discovered. The nature of the spacers in fluroquinolone and
aminoglycoside parts greatly influenced the antibacterial activity.
The hybrids were significantly more potent than the parent
NeoB, especially against Gram-negative bacteria and Gram-
positive MRSA, and overcame most prevalent types of resistance
associated with aminoglycosides. The hybrids inhibited bacterial
protein synthesis with potencies similar to or better than that
of NeoB and were up to 32-fold more potent inhibitors than
Cipro for the fluoroquinolone targets, DNA gyrase, and TopoIV.
One case study also demonstrated a significant delay of
resistance formation in both Gram-negative (E. coli) and Gram-
positive (B. subtilis) bacteria to the treatment with Cipro-NeoB
hybrid in comparison to that of each drug (Cipro and NeoB)
separately or their 1:1 mixture.
Experimental Section
General Methods. 1H NMR spectra (including DEPT, 2D
COSY, 2D TOCSY, 1D TOCSY, HMQC, HMBC) were rou-
tinely recorded on a Bruker Avance 500 spectrometer, and
chemical shifts reported (in ppm) are relative to internal Me4Si
(δ ) 0.0) with CDCl3 as the solvent and to HOD (δ ) 4.63)
with D2O as the solvent. 13C NMR spectra were recorded on a
Bruker Avance 500 spectrometer at 125.8 MHz, and the chemical
shifts were reported (in ppm) relative to the residual solvent
signal for CDCl3 (δ ) 77.00) or to external sodium 2,2-dimethyl-

Fluoroquinolone-Aminoglycoside Hybrid Antibiotics
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2249
Figure 2. Representative comparative data for the inhibition of DNA gyrase (A and B) and TopoIV (C and D) with Cipro and compound 1f. (A)
A 1% agarose gel shows the inhibitory activity of 1f against DNA gyrase: lane 1, relaxed DNA; lane 2, supercoiling reaction by DNA gyrase
without presence of inhibitor; lanes 3-8, same as lane 1 but in the presence of 30, 60, 100, 150, 200, and 300 nM inhibitor 1f. (B) Semilogarithmic
plot of in vitro DNA gyrase supercoiling reaction inhibition, measured for Cipro and 1f. (C) A 1% agarose gel shows the inhibitory activity of 1f
against TopoIV: lane 1, supercoiled DNA; lane 2, relaxation reaction by TopoIV without the presence of inhibitor; lanes 3-8, same as lane 1 but
in the presence of 0.2, 0.3, 0.5, 0.8, 1.2, and 10 µM 1f. (D) Semilogarithmic plot of TopoIV inhibition, measured for Cipro and 1f. The percentages
of the supercoiled DNA were calculated from the electrophoresis images by using ImageJ Launcher program (Rasband, W. Bethesda, MD) and
plotted as functions of drug concentration. Each data point represents the average of two to three independent experimental results.
Figure 3. Comparative study on the emergence of resistance in E. coli and B. subtilis after 15 serial passages in the presence of Cipro, NeoB,
Cipro + NeoB mixture (1:1 molar ratio), and hybrid structure 1i. Relative MIC is the normalized ratio of MIC obtained for a given subculture to
MIC obtained upon first exposure.
2-silapentane sulfonate (δ ) 0.0) for D2O as the solvent. Mass
spectra were obtained either on a Bruker Daltonix Apex 3 mass
spectrometer under electron spray ionization (ESI) or by a TSQ-
70B mass spectrometer (Finnigan Mat). Reactions were moni-
tored by TLC on silica gel 60 F254 (0.25 mm, Merck), and spots
were visualized by charring with a yellow solution containing
(NH4)Mo7O24 · 4H2O (120 g) and (NH4)2Ce(NO3)6 (5 g) in 10%
H2SO4 (800 mL). Flash column chromatography was performed
on silica gel 60 (70-230 mesh). All reactions were carried out
under an argon atmosphere with anhydrous solvents, unless
otherwise noted. IR spectra (CHCl3) were recorded on a Bruker
vector 22 spectrophotometer, and only significant peaks were
identified. Microwave assisted reactions were carried out in
domestic microwave oven, Sauter SG251. Analytical HPLC was
performed on Hitachi LC system equipped with autosampler,
by using Superspher 100 RP-18 column and a detection
wavelength of 271 nm. All chemicals, unless otherwise stated,
were obtained from commercial sources. 1-Bromo-2-chloroet-
hane, 1-bromo-3-chloropropane, 1-bromo-4-chlorobutane, 1-bromo-
5-chloropentane, 1,6-dibromohexane, 1,3-dibromo-2-propanol,
2-bromoethyl ether, R,R′-dibromo-m-xylene, and R,R′-dibromo-
p-xylene as well as 4-ethynylaniline and propargylamine were
obtained from Sigma-Aldrich Israel. Compound 7 was prepared
as previously reported.30 Purity of the hybrids 1a-q were
determined by using HPLC analysis which indicated >95% purity
of each product (see Supporting Information).
General Procedure for Preparation of 2a-i. A mixture of
ciprofloxacin (1 mmol) and the azido linker (5 mmol) in acetonitrile
(15 mL) was refluxed in the presence of powdered NaHCO3 (1
mmol) for 12-24 h. When TLC (MeOH/CH2Cl2, 1:9) indicated
completion of the reaction (2-14 h), the mixture was filtered and
washed with excess MeOH/CH2Cl2 (1:1) and the combined filtrates
were evaporated to dryness. The residue was purified by column
chromatography (silica gel, MeOH/CH2Cl2, 1:10) to yield the
product usually as a slightly yellow solid.

2250 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
PokroVskaya et al.
7-(4-(2-Azidoethyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxylic Acid (2a). 1H NMR (500
MHz, CDCl3) δH 1.14-1.15 (d, J ) 3.0 Hz, 2H, cyclopropane),
1.34-1.35 (d, J ) 7.0 Hz, 2H, cyclopropane), 2.65-2.67 (t, J )
6.0 Hz, 2H, NCH2), 2.69-2.71 (t, J ) 5.0 Hz, 4H, piperazine),
3.31-3.33 (t, J ) 5.0 Hz, 4H, piperazine), 3.34-3.36 (t, J ) 6.0
Hz, 2H, CH2N3), 3.51-3.53 (m, 1H, cyclopropane), 7.29-7.30 (d,
J ) 7.0 Hz, 1H, C8-H), 7.78-7.80 (d, J ) 14.0 Hz, 1H, C5-H),
8.61 (s, 1H, C2-H). 13C NMR (125 MHz, CDCl3) δC 9.9 (CH2 of
cyclopropane), 10.0 (CH2 of cyclopropane), 31.5 (CH of cyclo-
propane), 49.8 (CH2N3), 51.4, 54.5, 58.9 (NCH2), 106.7, 109.6,
113.7, 121.2, 140.8, 147.6, 149.1, 154.4, 156.4, 168.8, 178.7. IR
(CHCl3, cm-1): 2120 (N3), 1730 (CO). MALDI TOF MS calculated
for C19H21FN6O3Na ([M + Na]+) m/e 423.4; measured m/e 423.2.
7-(4-(3-Azidopropyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-
4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (2b). 1H NMR
(500 MHz, CDCl3) δH 1.19 (m, 2H, cyclopropane), 1.38-1.39 (d,
J ) 6.0 Hz, 2H, cyclopropane), 1.79-1.84 (m, 2H, CH2 of linker),
2.52-2.54 (t, J ) 7.0 Hz, 2H, NCH2), 2.68 (m, 4H, piperazine),
3.36 (m, 4H, piperazine), 3.38-3.40 (t, J ) 7.0 Hz, 2H, CH2N3),
3.54-3.55 (m, 1H, cyclopropane), 7.34-7.35 (d, J ) 7.0 Hz, 1H,
C8-H), 7.91-7.93 (d, J ) 7.0 Hz, 1H, C5-H), 8.70 (s, 1H, C2-H).
13C NMR (125 MHz, CDCl3) δC 10.0 (CH2 of cyclopropane), 28.0
(CH2 of linker), 37.1 (CH of cyclopropane), 51.2, 51.5 (CH2N3),
54.5, 56.8 (NCH2), 106.6, 109.7, 113.9, 114.1, 121.4, 140.9, 147.6,
149.1, 154.4, 156.4, 168.8, 178.8. IR (CHCl3, cm-1): 2100 (N3),
1722 (CO). MALDI TOF MS calculated for C20H23FN6O3Na ([M
+ Na]+) m/e 437.4; measured m/e 437.4.
7-(4-(4-Azidobutyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxylic Acid (2c). 1H NMR (500
MHz, CDCl3) δH 1.17 (m, 2H, cyclopropane), 1.36-1.37 (d, J )
6.0 Hz, 2H, cyclopropane), 1.62-1.65 (m, 4H, CH2 of linker),
2.44-2.46 (t, J ) 7.0 Hz, 2H, NCH2), 2.65 (m, 4H, piperazine),
3.31-3.34 (m, 4H, piperazine; 2H, CH2N3), 3.54-3.55 (m, 1H,
cyclopropane), 7.31-7.33 (d, J ) 7.0 Hz, 1H, C8-H), 7.82-7.85
(d, J ) 7.0 Hz, 1H, C5-H), 8.64 (s, 1H, C2-H). 13C NMR (125
MHz, CDCl3) δC 10.0 (CH2 of cyclopropane), 25.7 (CH2 of linker),
28.6 (CH2 of linker), 37.1 (CH of cyclopropane), 51.2, 51.5, 53.1
(CH2N3), 54.5, 59.5 (NCH2), 106.6, 109.6, 113.7, 113.9, 121.2,
140.8, 147.6, 149.1, 154.4, 156.4, 168.7, 178.7. IR (CHCl3, cm-1):
2100 (N3), 1718 (CO). MALDI TOF MS calculated for
C21H25FN6O3 ([M + H]+) m/e 429.5; measured m/e 429.4.
7-(4-(5-Azidopentyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxylic Acid (2d). 1H NMR (500
MHz, CDCl3) δH 1.20 (m, 2H, cyclopropane), 1.38-1.40 (d, J )
7.0 Hz, 2H, cyclopropane), 1.42-1.47 (m, 2H, CH2 of linker),
1.55-1.61 (m, 2H, CH2 of linker), 1.62-1.68 (m, 2H, CH2 of
linker), 2.44-2.47 (t, J ) 7.0 Hz, 2H, NCH2), 2.68 (m, 4H,
piperazine), 3.28-3.31 (t, J ) 6.0 Hz, 2H, CH2N3), 3.36 (m, 4H,
piperazine), 3.54-3.56 (m, 1H, cyclopropane), 7.35-7.36 (d, J )
7.0 Hz, 1H, C8-H), 7.96-7.99 (d, J ) 13.0 Hz, 1H, C5-H), 8.75
(s, 1H, C2-H). 13C NMR (125 MHz, CDCl3) δC 10.0 (CH2 of
cyclopropane), 26.4 (CH2 of linker), 28.1 (CH2 of linker), 30.5(CH2
of linker), 37.1 (CH of cyclopropane), 51.6, 53.2 (CH2N3), 54.6,
60.0 (NCH2), 106.5, 109.9, 114.1, 114.2, 121.5, 140.9, 147.7, 154.5,
156.5, 168.9, 178.9. IR (CHCl3, cm-1): 2110 (N3), 1723 (CO).
MALDI TOF MS calculated for C22H27FN6O3 ([M + H]+) m/e
443.5; measured m/e 443.3.
(CO). MALDI TOF MS calculated for C23H29FN6O3 ([M + H]+)
m/e 457.3; measured m/e 457.5.
7-(4-(3-Azido-2-hydroxypropyl)piperazin-1-yl)-1-cyclopropyl-
1
6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (2f). H
NMR (500 MHz, CDCl3) δH 1.20-1.21 (d, J ) 4.0 Hz, 2H,
cyclopropane), 1.39-1.41 (d, J ) 7.0 Hz, 2H, cyclopropane),
2.46-2.49 (dd, J ) 3.0, 12.0 Hz, 1H, NCH2), 2.57-2.62 (dd, J )
10.0, 12.0 Hz, 1H, NCH2), 2.67-2.71 (m, 2H, piperazine),
2.87-2.92 (m, 2H, piperazine), 3.24-3.28 (dd, J1 ) 6.0 Hz,
J2)13.0 Hz, 1H, CH2N3), 3.36-3.39 (dd, J1 ) 6.0 Hz, J2)13.0
Hz, 4H, piperazine), 3.44-3.47 (dd, J1 ) 4.0 Hz, J2 ) 9.0 Hz, 1H,
CH2N3), 3.54-3.58 (m, 1H, cyclopropane), 3.94-3.98 (m, 1H,
CH-OH), 7.34-7.36 (d, J ) 7.0 Hz, 1H, C8-H), 7.91-7.94 (d,
J ) 13.0 Hz, 1H, C5-H), 8.70 (s, 1H, C2-H). 13C NMR (125 MHz,
CDCl3) δC 10.0 (CH2 of cyclopropane), 37.2 (CH of cyclopropane),
51.6, 54.7, 56.1, 62.4, 68.0, 106.7, 109.8, 114.0, 114.2, 121.5, 140.8,
147.5, 149.2, 154.4, 156.4, 168.8, 178.8. IR (CHCl3): 2110 (N3),
1723 (CO). MALDI TOF MS calculated for C20H23FN6O4 ([M +
H]+) m/e 431.4; measured m/e 431.0.
7-(4-(2-(2-Azidoethoxy)ethyl)piperazin-1-yl)-1-cyclopropyl-6-
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (2g). 1H
NMR (500 MHz, CDCl3) δH 1.17-1.20 (m, 2H, cyclopropane),
1.36-1.40 (m, 2H, cyclopropane), 2.72-2.74 (t, J ) 5.0 Hz, 2H,
NCH2), 2.78-2.79 (m, 4H, piperazine), 3.37-3.41 (m, 4H, pip-
erazine; 2H, CH2N3), 3.53-3.57 (m, 1H, cyclopropane), 3.66-3.68
(t, J ) 4.0 Hz, 2H, CH2 of linker), 3.69-3.71 (t, J ) 4.0 Hz, 2H,
CH2 of linker), 7.34-7.35 (d, J ) 8.0 Hz, 1H, C8-H), 7.92-7.95
(d, J ) 13.0 Hz, 1H, C5-H), 8.71 (s, 1H, C2-H). 13C NMR (125
MHz, CDCl3) δC 10.0 (CH2 of cyclopropane), 37.1 (CH of
cyclopropane), 51.4, 52.5 (CH2N3), 55.0, 59.5 (NCH2), 70.9 (CH2
of linker), 71.7 (CH2 of linker), 106.6, 109.8, 114.0, 114.1, 121.4,
140.9, 147.7, 149.1, 154.4, 156.4, 168.8, 178.8. IR (CHCl3, cm-1):
2110 (N3), 1722 (CO). MALDI TOF MS calculated for
C21H25FN6O4 ([M + H]+) m/e 445.3; measured m/e 445.5.
7-(4-(3-(Azidomethyl)benzyl)piperazin-1-yl)-1-cyclopropyl-6-
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (2h). 1H
NMR (500 MHz, CDCl3) δH 1.16-1.19 (m, 2H, cyclopropane),
1.34-1.38 (m, 2H, cyclopropane), 2.67-2.69 (t, J ) 4.5 Hz, 4H,
piperazine), 3.35-3.37 (t, J ) 4.5 Hz, 4H, piperazine), 3.51-3.55
(m, 1H, cyclopropane), 3.62 (s, 2H, NCH2), 4.35 (s, 2H, CH2N3),
7.22-7.24 (m, 1H, aromatic), 7.30-7.37 (m, 4H, aromatic, C8-H),
7.86-7.89 (d, J ) 13.0 Hz, 1H, C5-H), 8.67 (s, 1H, C2-H). 13C
NMR (125 MHz, CDCl3) δC 10.0 (CH2 of cyclopropane), 31.4 (CH
of cyclopropane), 51.5, 54.5, 56.5 (CH2N3), 64.4 (NCH2), 106.6,
109.7, 113.8, 114.0, 121.2, 129.0, 130.7, 130.9, 137.3, 140.3, 140.9,
154.4, 156.4, 168.8, 178.7. MALDI TOF MS calculated for
C25H25FN6O3 ([M + H]+) m/e 477.2; measured m/e 477.5.
7-(4-(4-(Azidomethyl)benzyl)piperazin-1-yl)-1-cyclopropyl-6-
fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic Acid (2i). 1H
NMR (500 MHz, CDCl3) δH 1.17-1.20 (m, 2H, cyclopropane),
1.35-1.39 (m, 2H, cyclopropane), 2.68-2.69 (t, J ) 5.0 Hz, 4H,
piperazine), 3.35-3.37 (t, J ) 5 Hz, 4H, piperazine), 3.52-3.54
(m, 1H, cyclopropane), 3.61 (s, 2H, NCH2), 4.34 (s, 2H, CH2N3),
7.29-7.31 (d, J ) 7.5 Hz, 2H, aromatic), 7.33-7.34 (d, J ) 7.0
Hz, 1H, C8-H), 7.38-7.39 (d, J ) 7.5 Hz, 2H, aromatic),
7.91-7.94 (d, J ) 13.0 Hz, 1H, C5-H), 8.70 (s, 1H, C2-H). 13C
NMR (125 MHz, CDCl3) δC 8.1 (CH2 of cyclopropane), 35.2 (CH
of cyclopropane), 49.7, 52.6 (CH2N3), 54.5, 62.4 (NCH2), 104.7,
107.9, 112.1, 112.3, 119.5, 128.2, 129.5, 134.4, 137.9, 139.0, 145.9,
147.3, 152.6, 154.6, 167.0, 177.0. MALDI TOF MS calculated for
C25H25FN6O3 ([M + H]+) m/e 477.1; measured m/e 477.5.
1,3,2′,6′,2′′′,6′′′-Hexaazido-5′′-triisopropylsilyloxyneomycin (4).
Commercial NeoB was converted to the corresponding perazido
derivative according to the published procedure.29 Hexaazido-NeoB
(5.10 g, 6.62 mmol) was dissolved in pyridine (30 mL), and
4-DMAP (cat.) was added. The mixture was stirred at room
temperature. After 15 min, triisopropylsilylchloride (TIPSCl) (1.91
g, 9.93 mmol) was added, and TLC (EtOAc, 100%) indicated
completion after 3 h. The mixture was diluted with EtOAc and
washed with brine, H2SO4 (2%), NaHCO3 (sat.), and brine. Organic
layers were combined, dried over MgSO4, evaporated and the
7-(4-(6-Azidohexyl)piperazin-1-yl)-1-cyclopropyl-6-fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxylic Acid (2e). 1H NMR (500
MHz, CDCl3) δH 1.18-1.19 (m, 2H, cyclopropane), 1.36-1.43 (m,
2H, cyclopropane; 4H, CH2 of linker), 1.54-1.63 (m, 4H, CH2 of
linker), 2.42-2.45 (t, J ) 7.5 Hz, 2H, NCH2), 2.68 (m, 4H,
piperazine), 3.26-3.28 (t, J ) 6.5 Hz, 2H, CH2N3), 3.35-3.37 (m,
4H, piperazine), 3.53-3.57 (m, 1H, cyclopropane), 7.33-7.35 (d,
J ) 7.0 Hz, 1H, C8-H), 7.90-7.93 (d, J ) 13.5 Hz, 1H, C5-H),
8.70 (s, 1H, C2-H). 13C NMR (125 MHz, CDCl3) δC 10.0 (CH2
cyclopropane), 28.4 (CH2 of linker), 28.8 (CH2 of linker), 30.6 (CH2
of linker), 37.1 (CH of cyclopropane), 51.5, 53.2 (CH2N3), 54.6,
60.1 (NCH2), 106.5, 109.8, 113.9, 114.1, 121.4, 140.9, 147.7, 149.1,
154.4, 156.4, 168.8, 178.8. IR (CHCl3, cm-1): 2110 (N3), 1726

Fluoroquinolone-Aminoglycoside Hybrid Antibiotics
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2251
residue was purified by flash chromatography (silica gel, EtOAc/
hexane) to yield the silyl ether 4 as a white powder (4.60 g, 75%
yield). 1H NMR (500 MHz, CDCl3) δH 0.85-0.98 (m, 15H, TIPS);
ring I δH 3.14-3.28 (m, 3H, H-2, H-4, H-6), 3.33-3.51 (m, 1H,
H-6′), 3.63-3.74 (m, 1H, H-3), 3.91-3.95 (m, 1H, H-5), 5.65-5.66
(d, J ) 3.5 Hz, 1H, H-1); ring II δH 1.19-1.24 (ddd, J1 ) J2 ) J3
) 12.5 Hz, 1H, H-2ax), 2.01-2.06 (dt, J ) 4.0, 12.5 Hz, 1H,
H-2eq), 3.14-3.28 (m, 2H, H-1, H-3), 3.33-3.51 (m, 3H, H-4,
H-5, H-6); ring III δH 3.33-3.51 (m, 1H, H-5′), 3.63-3.74 (m,
1H, H-5), 4.03-4.05 (m, 2H, H-2, H-4), 4.22-4.23 (dd, J ) 4.0,
4.5 Hz, 1H, H-3), 5.10-5.11 (d, J ) 4.5 Hz, 1H, H-1); ring IV δH
2.91-2.94 (dd, J ) 4.0, 10.5 Hz, 1H, H-6), 3.14-3.28 (m, 2H,
H-4, H-6), 3.63-3.74 (m, 1H, H-2), 3.78-3.80 (t, J ) 3.5 Hz,
1H, H-3), 3.81-3.83 (m, 1H, H-5), 4.96 (d, J ) 1.0 Hz, 1H, H-1).
13C NMR (125 MHz, CDCl3) δC 11.5, 17.4, 31.6 (C-2), 50.8 (C-
6′), 51.1 (C-6′′′), 59.2, 59.4, 60.4, 63.2, 63.3, 68.4, 68.7, 70.5, 70.9,
71.1, 73.6, 74.3, 75.0, 75.3, 76.3, 83.1, 85.1, 96.3 (C-1′′′), 98.6
(C-1′), 107.2 (C-1′′). MALDI TOF MS calcd for C32H54N18O13SiNa
([M + Na]+) m/e 949.6; measured m/e 949.4.
1,3,2′,6′,2′′′,6′′′-Hexaazido-6,3′,4′,2′′,3′′′,4′′′-hexa(4-methoxy-
benzyloxy)-5′′-triisopropylsilyloxyneomycin (5). Compound 4 (3.0
g, 3.24 mmol) was dissolved in anhydrous DMF (20 mL), and after
the mixture was stirred at 0 °C for 10 min 4-methoxybenzyl chloride
(4.6 g, 29.2 mmol) and NaH (0.17 g, 7.29 mmol) were added. The
mixture was allowed to warm to room temperature, and after 4 h
TLC (EtOAc, 100%) indicated completion. The mixture was diluted
with EtOAc and extensively washed with brine. The combined
organic layer was dried over MgSO4, evaporated to dryness, and
used in the next step without further purification.
(500 MHz, CDCl3) δH 2.44-2.45 (t, J ) 2.5 Hz, 1H, CH of triple
bond), 3.73 (s, 3H, CH3O), 3.78 (s, 3H, CH3O), 3.80 (s, 3H, CH3O),
3.81 (s, 3H, CH3O), 3.82 (s, 6H, CH3O), 4.13-4.14 (t, J ) 2.5
Hz, 2H, CH2 of linker), 4.39-4.43 (m, 4H, CH2 of PMB),
4.49-4.66 (m, 4H, CH2 of PMB), 4.77-4.89 (m, 4H, CH2 of PMB),
6.72-6.74 (m, 2H, aromatic), 6.84-6.89 (m, 10H, aromatic),
7.12-7.31 (m, 12H, aromatic); ring I δH 3.28-3.32 (m, 1H, H-6),
3.35-3.37 (dd, J ) 3.5, 10.5 Hz, 1H, H-2), 3.41-3.51 (m, 1H,
H-4), 3.56-3.58 (dd, J ) 3.0, 10.0 Hz, 1H, H-6′), 4.02-4.06 (dd,
J ) 9.5, 10.0 Hz, 1H, H-3), 4.24-4.27 (m, 1H, H-5), 6.08-6.09
(d, J ) 3.5 Hz, 1H, H-1); ring II δH 1.39-1.46 (ddd, J1 ) J2 ) J3
) 12.5 Hz, 1H, H-2ax), 2.22-2.27 (dt, J ) 4.0, 12.5 Hz, 1H,
H-2eq), 3.28-3.32 (m, 1H, H-1), 3.41-3.51 (m, 2H, H-3, H-5),
3.63-3.67 (t, J ) 9.0 Hz, 1H, H-4), 3.89-3.92 (t, J ) 9.0 Hz, 1H,
H-6); ring III δH 3.41-3.51 (m, 1H, H-5), 3.67-3.72 (m, 2H, H-2,
H-5′), 4.16-4.18 (m, 1H, H-4), 4.24-4.27 (m, 1H, H-3), 5.61-5.62
(d, J ) 5.5 Hz, 1H, H-1); ring IV δH 2.86-2.91 (dd, J ) 4.0, 13.0
Hz, 1H, H-6), 3.09 (m, 1H, H-2), 3.41-3.51 (m, 1H, H-4),
3.67-3.73 (m, 3H, H-3, H-5, H-6′), 4.95 (d, J ) 1.5 Hz, 1H, H-1).
13C NMR (125 MHz, CDCl3) δC 30.3, 32.4 (C-2), 51.2 (C-6′), 55.2
(C-6′′), 57.4, 58.5, 59.8, 60.4, 63.2, 69.5, 70.9, 71.0, 71.2, 71.9,
72.4, 73.0, 74.2, 74.3, 74.5, 75.0, 75.2, 78.1, 79.5, 79.7, 81.6, 82.1,
83.8, 96.2 (C-1′′′), 98.2 (C-1′), 107.1 (C-1′′), 113.7, 113.8, 114.0,
129.4, 129.5 L, 129.8, 130.0, 159.1, 159.2, 159.5. MALDI TOF
MS calcd for C74H84N18O19Na ([M + Na]+) m/e 1551.8; measured
m/e 1551.4.
5′′-(Prop-2-ynyloxy)neomycin (3a). Compound 6 (0.43 g, 0.28
mmol) was dissolved in acetonitrile (5 mL), and after the mixture
was stirred at -4 °C for 10 min, cerium(IV) ammonium nitrate
(CAN) (1.0 g, 1.82 mmol) in 0.5 mL of water was added. The
reaction progress was monitored by TLC (EtOAc/hexane, 4:5, and
EtOAc, 100%). After 3 h, the reaction mixture was diluted with
EtOAc (100 mL) and washed with brine. The combined organic
layer was dried over MgSO4, evaporated to dryness, and used in
the next step without further purification.
The crude from the previous step was dissolved in dry THF (10
mL), cooled at 0 °C, and 1 M solution of tetrabutyl ammonium
fluoride in THF (3.74 mL, 3.74 mmol) was added. The reaction
progress was monitored by TLC (EtOAc/hexane, 2:3), which
indicated completion after 3 h. The mixture was diluted with EtOAc
(300 mL) and washed with brine. The combined organic layer was
dried over MgSO4, evaporated, and purified by flash chromatog-
raphy (silica gel, EtOAc/hexane) to yield 5 (3.5 g, 72% for two
The crude product from the previous step was dissolved in THF
(7 mL), 0.1 M NaOH (1.5 mL) and stirred at 60 °C for 10 min,
after which PMe3 (1 M solution in THF, 4.0 mL, 4.0 mmol) was
added. The reaction progress was monitored by TLC (CH2Cl2/
MeOH/H2O/MeNH2 (33% solution in EtOH), 10:15:6:15), which
indicated completion after 3.5 h. The reaction mixture was purified
by flash chromatography on a short column of silica gel, and the
column was washed as follows: THF, EtOH, MeOH, and finally
with MeNH2 (33% solution in EtOH). The fractions containing the
product were combined and evaporated to dryness, redissolved in
water, and evaporated again to afford the product 3a as a free amine
(162.5 mg, 89% for two steps). This product was then dissolved in
water, the pH was adjusted to 7.5 with 0.01 M H2SO4, and the
mixture was lyophilized to give the sulfate salt of 3a (220 mg) as
1
steps). H NMR (500 MHz, CDCl3) δH 3.74 (s, 3H, CH3O), 3.78
(s, 3H, CH3O), 3.80 (s, 6H, CH3O), 3.82 (s, 6H, CH3O), 4.30-4.41
(m, 2H, CH2 of PMB), 4.50-4.58 (m, 4H, CH2 of PMB), 4.64-4.67
(m, 4H, CH2 of PMB), 4.78-4.87 (m, 2H, CH2 of PMB), 6.75-6.76
(m, 2H, aromatic), 6.86-6.90 (m, 10H, aromatic), 7.13-7.35 (m,
12H, aromatic); ring I δH 3.12-3.15 (dd, J ) 4.0, 10.0 Hz, 1H,
H-2), 3.29-3.36 (m, 1H, H-6), 3.40-3.51 (m, 2H, H-4, H-6′),
4.03-4.07 (dd, J ) 9.0, 11.0 Hz, 1H, H-3), 4.21-4.23 (m, 1H,
H-5), 5.88-5.89 (d, J ) 4.0 Hz, 1H, H-1); ring II δH 1.41-1.49
(ddd, J1 ) J2 ) J3 ) 12.5 Hz, 1H, H-2ax), 2.26-2.35 (dt, J ) 4.0,
12.5 Hz, 1H, H-2eq), 3.29-3.36 (m, 1H, H-1), 3.40-3.51 (m, 2H,
H-3, H-5), 3.62-3.66 (t, J ) 10.0 Hz, 1H, H-4), 3.90-3.94 (t, J
) 9.0 Hz, 1H, H-6); ring III δH 3.02-3.04 (dd, J ) 6.0, 9.0 Hz,
1H, H-5), 3.67-3.77 (m, 2H, H-2, H-5′), 4.11-4.12 (m, 1H, H-4),
4.33-4.35 (dd, J ) 4.0, 6.0 Hz, 1H, H-3), 5.68-5.69 (d, J ) 5.0
Hz, 1H, H-1); ring IV δH 2.97-3.01 (dd, J ) 4.0, 12.0 Hz, 1H,
H-6), 3.12 (m, 1H, H-4), 3.40-3.51 (m, 1H, H-2), 3.67-3.77 (m,
3H, H-3, H-5, H-6′), 4.97 (d, J ) 2.0 Hz, 1H, H-1). 13C NMR
(125 MHz, CDCl3) δC 32.3 (C-2), 51.1 (C-6′), 55.2 (C-6′′), 57.4,
59.6, 60.4, 61.9, 62.6, 71.0, 71.3, 72.0, 72.5, 72.9, 74.2, 74.6, 74.9,
75.7, 78.2, 78.6, 80.8, 81.9, 83.0, 83.9, 97.1 (C-1′′′), 98.9 (C-1′),
105.4 (C-1′′). MALDI TOF MS calcd for C71H82N18O19Na ([M +
Na]+) m/e 1513.7; measured m/e 1513.5.
1
a white foamy solid. H NMR (500 MHz, D2O, pH 3.0) δH 2.96
(m, 1H, CH of triple bond), 4.23-4.28 (m, 2H, CH2 of linker);
ring I δH 3.27-3.51 (m, 4H, H-2, H-4, H-6, H-6′), 3.82-3.93 (m,
2H, H-3, H-5), 6.07-6.08 (d, J ) 4.0 Hz, 1H, H-1); ring II δH
1.96-2.03 (ddd, J1 ) J2 ) J3 ) 12.5 Hz, 1H, H-2ax), 2.37-2.41
(dt, J ) 4.0, 12.5 Hz, 1H, H-2eq), 3.27-3.51 (m, 2H, H-1, H-3),
3.64-3.69 (m, 1H, H-5), 4.00-4.04 (t, J ) 9.5 Hz, 1H, H-6),
4.14-4.17 (t, J ) 9.5 Hz, 1H, H-4); ring III δH 3.64-3.69 (m, 1H,
H-5), 3.82-3.93 (m, 1H, H-5′), 4.23-4.28 (m, 1H, H-4), 4.40 (dd,
J ) 2.0, 4.0 Hz, 1H, H-2), 4.47-4.49 (dd, J ) 4.0, 7.0 Hz, 1H,
H-3), 5.36 (s, 1H, H-1); ring IV δH 3.08-3.13 (dd, J ) 8.5, 13.5
Hz, 1H, H-6), 3.27-3.51 (m, 2H, H-2, H-6′), 3.75 (m, 1H, H-4),
4.14-4.17 (m, 1H, H-3), 4.23-4.28 (m, 1H, H-5), 5.22 (s, 1H,
H-1). 13C NMR (125 MHz, D2O) δC 29.6 (C-2), 42.2 (C-6′), 42.5
(C-6′′′), 50.2, 51.6, 52.6, 55.3, 60.3, 68.9, 69.4, 69.6, 71.3, 72.1,
73.2, 74.1, 74.9, 76.2, 77.2, 78.9, 81.1(CH of triple bond), 81.6,
87.1, 96.4 (C-1′′′), 96.9 (C-1′), 112.5 (C-1′′). MALDI TOF MS
calcd for C26H49N6O13 ([M + H]+) m/e 653.3; measured m/e 653.3.
1,3,2′,6′,2′′′,6′′′-Hexaazido-6,3′,4′,2′′,3′′′,4′′′-hexaacetoxyneo-
mycin-4′′-carboxylic Acid (8). Compound 730 (0.43 g, 0.42 mmol)
was dissolved in CH2Cl2 (30 mL) and cooled to 5 °C. Then water
(2.5 mL), TEMPO (0.013 g, 0.08 mmol), and BAIB (0.34 g, 1.06
1,3,2′,6′,2′′′,6′′′-Hexaazido-6,3′,4′,2′′,3′′′,4′′′-hexa(4-methoxy-
benzyloxy)-5′′-(prop-2-ynyloxy)neomycin (6). Compound 5 (0.73
g, 0.49 mmol) was dissolved in dry DMF (15 mL) and was stirred
at 0 °C for 10 min, followed by the addition of tetrabutylammonium
iodide (0.54 g, 1.47 mmol), propargyl bromide (0.35 g, 2.97 mmol),
and NaH (0.070 g, 2.94 mmol). The reaction progress was
monitored by TLC (EtOAc/hexane, 4:5), which indicated comple-
tion after 4 h. The mixture was diluted with EtOAc (200 mL) and
washed with brine. The combined organic layer was dried over
MgSO4, evaporated, and purified by flash chromatography (silica
gel, EtOAc/hexane) to yield compound 6 (0.60 g, 80%). 1H NMR

2252 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
PokroVskaya et al.
mmol) were added. The reaction mixture was stirred at 5 °C for
40 min and then allowed slowly to warm to room temperature.
The reaction progress was monitored by TLC with two solvent
systems (EtOAc/hexane, 1:1, and MeOH/CHCl3, 1:9), which
indicated completion after 4.5 h. The mixture was cooled to 0 °C,
diluted with EtOAc, quenched with Na2S2O3, and washed with
brine. The combined organic layer was dried over MgSO4,
evaporated, and purified by flash chromatography (silica gel, MeOH/
CHCl3) to yield 8 (320 mg, 76%). 1H NMR (500 MHz, CDCl3) δH
2.04 (s, 3H, OAc), 2.06 (s, 3H, OAc), 2.12 (s, 3H, OAc), 2.13 (s,
3H, OAc), 2.15 (s, 3H, OAc), 2.16 (s, 3H, OAc); ring I δH
3.27-3.44 (m, 1H, H-2), 3.50-3.57 (m, 2H, H-6, H-6′), 4.41-4.43
(m, 1H, H-5), 5.00 (t, J ) 9.5 Hz, 1H, H-4), 5.43 (t, J ) 10.5 Hz,
1H, H-3), 6.12 (s, 1H, H-1); ring II δH 1.63 (ddd, J1 ) J2 ) J3 )
12.5 Hz, 1H, H-2ax), 2.37 (dt, J ) 3.5, 12.5 Hz, 1H, H-2eq),
3.27-3.44 (m, 2H, H-1, H-3), 3.73 (t, J ) 9.0 Hz, 1H, H-5), 3.96
(t, J ) 9.0 Hz, 1H, H-4), 4.93-4.97 (m, 1H, H-6); ring III δH 4.71
(m, 1H, H-3), 4.81 (t, J ) 5.0 Hz, 1H, H-2), 4.87 (d, J ) 3.0 Hz,
1H, H-4), 5.54 (d, J ) 5.0 Hz, 1H, H-1); ring IV δH 3.27-3.44
(m, 3H, H-2, H-6, H-6′), 4.05-4.07 (m, 1H, H-5), 4.71 (m, 1H,
H-4), 4.95 (s, 1H, H-1), 5.05 (t, J ) 2.5 Hz, 1H, H-3). 13C NMR
(125 MHz, CDCl3) δC 20.4, 20.7, 20.8, 21.0, 31.2 (C-2), 50.5 (C-
6′′′), 50.9 (C-6′), 57.2, 58.0, 59.0, 60.7, 65.7, 68.6, 69.1, 69.3, 69.8,
73.0, 74.6, 75.2, 76.0, 79.3, 81.8, 96.5 (C-1′), 100.3 (C-1′′′), 106.3
(C-1′′), 168.6, 169.7, 169.8, 170.1, 170.2. MALDI TOF MS calcd
for C35H44N18O20K ([M + K]+) m/e 1075.3; measured m/e 1075.4.
column of silica gel, and the column was washed as follows: THF,
EtOH, MeOH, and finally MeNH2 (33% solution in EtOH). The
fractions containing the product were evaporated under vacuum,
redissolved in water, and evaporated again to afford the product
3b as a free amine (1.39 g, 92%). This product was then dissolved
in water, the pH was adjusted to 7.5 with 0.01 M H2SO4, and the
mixture was lyophilized to give the sulfate salt of 3b (1.88 g) as a
white foamy solid. 1H NMR (500 MHz, D2O, pH 3.17) δH
2.59-2.60 (t, J ) 2.5 Hz, 1H, CH of triple bond), 3.86-4.02 (m,
2H, CH2 of linker); ring I δH 3.20-3.24 (dd, J ) 3.5, 13.5 Hz, 1H,
H-6), 3.25-3.37 (m, 3H, H-2, H-4, H-6′), 3.86-4.02 (m, 2H, H-3,
H-5), 6.03-6.04 (d, J ) 4.0 Hz, 1H, H-1); ring II δH 1.92-1.98
(ddd, J1 ) J2 ) J3 ) 12.5 Hz, 1H, H-2ax), 2.37-2.41 (dt, J ) 4.0,
12.5 Hz, 1H, H-2eq), 3.25-3.37 (m, 1H, H-1), 3.45-3.50 (m, 1H,
H-3), 3.67-3.71 (m, 1H, H-5), 3.86-4.05 (m, 1H, H-6), 4.15-4.19
(t, J ) 9.5 Hz, 1H, H-4); ring III δH 4.42-4.43 (dd, J ) 2.0, 4.5
Hz, 1H, H-3), 4.47-4.48 (d, J ) 7.5 Hz, 1H, H-4), 4.60-4.62
(dd, J ) 4.5, 7.5 Hz, 1H, H-2), 5.44 (s, 1H, H-1); ring IV δH
3.13-3.17 (dd, J ) 8.0, 13.5 Hz, 1H, H-6), 3.38-3.41 (dd, J )
3.0, 13.5 Hz, 1H, H-6′), 3.53 (m, 1H, H-2), 3.67-3.71 (m, 1H,
H-4), 4.15-4.19 (m, 1H, H-3), 4.20-4.23 (m, 1H, H-5), 5.20 (s,
1H, H-1). 13C NMR (125 MHz, D2O) δC 29.6 (C-2), 30.7, 42.1
(C-6′), 42.2 (C-6′′′), 50.2, 51.8, 52.5, 55.4, 68.7, 69.3, 69.5, 71.4,
72.5, 72.9, 74.0, 74.7, 76.4, 79.4, 80.9, 81.2 (CH of triple bond),
86.4, 96.5 (C-1′′′), 97.0 (C-1′), 112.3 (C-1′′), 173.0 (CO). MALDI
TOF MS calcd for C26H47N7O13K ([M + K]+) m/e 704.2; measured
m/e 704.3.
1,3,2′,6′,2′′′,6′′′-Hexaazido-6,3′,4′,2′′,3′′′,4′′′-hexaacetoxy-4′′-(4-
ethynylphenylcarbamoyl)neomycin (9b). The titled compound
was prepared as was described for the preparation of compound
9a with the following quantities: compound 8 (1.67 g, 1.61 mmol),
DCC (0.33 g, 1.61 mmol), HOBT (0.22 g, 1.61 mmol), 4-ethyny-
laniline (0.57 g, 4.8 mmol), DCM (20 mL). Yield: 1.57 g (86%).
1H NMR (300 MHz, CDCl3) δH 2.00 (s, 3H, OAc), 2.02 (s, 3H,
OAc), 2.09 (s, 3H, OAc), 2.10 (s, 3H, OAc), 2.11 (s, 3H, OAc),
2.15 (s, 3H, OAc), 3.00 (s, 1H, CH of triple bond), 7.37-7.40 (m,
2H, aromatic), 7.47-7.50 (m, 2H, aromatic), 8.60 (s, 1H, NH);
ring I δH 3.09-3.18 (m, 2H, H-2, H-6), 3.25-3.26 (m, 1H, H-6′),
4.38-4.44 (m, 1H, H-5), 5.03-5.04 (m, 1H, H-4), 5.39-5.45 (dd,
J ) 9.0, 10.5 Hz, 1H, H-3), 5.92-5.93 (d, J ) 4.0 Hz, 1H, H-1);
ring II δH 1.91-1.94 (ddd, J1 ) J2 ) J3 ) 12.5 Hz, 1H, H-2ax),
2.34-2.38 (dt, J ) 4.0, 12.5 Hz, 1H, H-2eq), 3.31-3.53 (m, 2H,
H-1, H-3), 3.66-3.72 (t, J ) 9.0 Hz, 1H, H-4), 3.94-4.00 (t, J )
9.0 Hz, 1H, H-5), 4.91-4.97 (t, J ) 9.0 Hz, 1H, H-6); ring III δH
4.64-4.75 (m, 2H, H-2, H-3), 4.78-4.80 (d, J ) 4.2 Hz, 1H, H-4),
5.53-5.55 (d, J ) 5.0 Hz, 1H, H-1); ring IV δH 3.25-3.26 (m,
1H, H-2), 3.31-3.54 (m, 2H, H-6, H-6′), 4.04-4.08 (m, 1H, H-5),
4.64-4.75 (m, 2H, H-3, H-4), 5.04 (s, 1H, H-1). 13C NMR (125
MHz, CDCl3) δC 22.1, 22.5, 22.7, 22.8, 33.0 (C-2), 51.0, 52.2 (C-
6′), 52.7 (C-6′′), 59.1, 59.8, 60.7, 62.2, 67.4, 70.4, 71.0, 71.1, 71.3,
74.9, 75.9, 76.9, 78.2, 78.9, 79.9, 82.7, 83.8, 85.0 (CH of triple
bond), 98.9 (C-1′), 101.8 (C-1′′′), 107.3 (C-1′′), 120.1, 121.7, 134.7,
139.6, 168.5, 170.4, 171.4, 171.5, 171.8, 171.9. MALDI TOF MS
calcd for C43H49N19O19Na ([M + Na]+) m/e 1158.3; measured m/e
1158.2.
1,3,2′,6′,2′′′,6′′′-Hexaazido-6,3′,4′,2′′,3′′′,4′′′-hexaacetoxy-4′′-
(prop-2-ynylcarbamoyl)neomycin (9a). Compound 8 (2.68 g, 2.59
mmol) was dissolved in CH2Cl2 (35 mL). Then DCC (0.53 g, 2.57
mmol) and HOBT (0.25 g, 1.85 mmol) were added at 0 °C. The
reaction mixture was stirred at 0 °C for 1 h and allowed slowly to
warm to room temperature. Then propargylamine (0.43 g, 7.81
mmol) was added. Progress of the reaction was monitored by TLC
with two solvent systems (EtOAc/hexane, 1:1, and MeOH/CHCl3,
1:9), which indicated completion after 4 h. The mixture was diluted
with EtOAc and washed with brine. The combined organic layer
was dried over MgSO4, evaporated, and purified by flash chroma-
1
tography (silica gel, EtOAc/hexane) to yield 9a (2.0 g, 72%). H
NMR (500 MHz, CDCl3) δH 2.07 (s, 3H, OAc), 2.10 (s, 3H, OAc),
2.13 (s, 6H, OAc), 2.17 (s, 3H, OAc), 2.19 (s, 3H, OAc), 2.25-2.26
(t, J ) 2.5 Hz, 1H, CH of triple bond), 3.91-3.95 (m, 1H, CH2 of
linker), 4.14-4.17 (m, 1H, CH2 of linker), 7.41-7.44 (t, J ) 6.0
Hz, 1H, NH); ring I δH 3.23-3.26 (dd, J ) 3.0, 10.0 Hz, 1H, H-2),
3.31-3.44 (m, 2H, H-6, H-6′), 4.46-4.49 (m, 1H, H-5), 4.99-5.09
(m, 1H, H-4), 5.50-5.54 (dd, J ) 9.0, 11.0 Hz, 1H, H-3),
5.97-5.98 (d, J ) 4.0 Hz, 1H, H-1); ring II δH 1.62-1.70 (ddd, J1
) J2 ) J3 ) 12.5 Hz, 1H, H-2ax), 2.39-2.43 (dt, J ) 4.0, 12.5
Hz, 1H, H-2eq), 3.31-3.44 (m, 1H, H-3), 3.54-3.58 (m, 1H, H-1),
3.74-3.78 (t, J ) 9.0 Hz, 1H, H-4), 3.99-4.02 (t, J ) 9.0 Hz, 1H,
H-5), 4.99-5.09 (m, 1H, H-6); ring III δH 4.57-4.60 (t, J ) 6.0
Hz, 1H, H-2), 4.65-4.66 (dd, J ) 3.5, 6.0 Hz, 1H, H-3), 4.82 (d,
J ) 4.0 Hz, 1H, H-4), 5.59-5.61 (d, J ) 6.0 Hz, 1H, H-1); ring
IV δH 3.31-3.44 (m, 2H, H-2, H-6), 3.54-3.58 (m, 1H, H-6′),
4.09-4.13 (m, 1H, H-5), 4.71-4.72 (t, J ) 2.0 Hz, 1H, H-4),
4.99-5.09 (m, 2H, H-1, H-3). 13C NMR (125 MHz, CDCl3) δC
22.1, 22.5, 22.7, 22.8, 30.6 (CH2 of linker), 33.2 (C-2), 52.4 (C-
6′), 52.7 (C-6′′′), 59.2, 59.9, 60.9, 62.0, 67.4, 70.6, 71.0, 71.1, 71.2,
73.5, 74.8, 75.8, 76.9, 77.7, 80.4 (CH of triple bond), 80.9, 83.1,
83.3, 99.0 (C-1′′′), 102.3 (C-1′), 106.7 (C-1′′), 170.1, 170.4, 171.4,
171.8, 171.9. MALDI TOF MS calcd for C38H47N19O19Na ([M +
Na]+) m/e 1096.3; measured m/e 1096.3.
4′′-(Prop-2-ynylcarbamoyl)neomycin (3b). Compound 9a (2.4
g, 2.27 mmol) was dissolved in 33% solution of MeNH2 in EtOH
(40 mL), and the mixture was stirred at room temperature for 30 h.
The reagent and the solvent were removed by evaporation, and the
residue was dissolved in THF (50 mL), NaOH 0.1 M (3 mL) and
stirred at 60 °C for 10 min, after which PMe3 (1 M solution in
THF, 21.9 mL, 21.9 mmol) was added. Propagation of the reaction
was monitored by TLC [CH2Cl2/MeOH/H2O/MeNH2 (33% solution
in EtOH), 10:15:6:15], which indicated completion after 3.5 h. The
reaction mixture was purified by flash chromatography on a short
4′′-(4-Ethynylphenylcarbamoyl)neomycin (3c). The titled com-
pound was prepared as was described for the preparation of 3b
with the following quantities: compound 9b (1.13 g, 1.00 mmol),
MeNH2 (70 mL), Me3P (1 M solution in THF, 8.86 mL, 8.86
mmol), NaOH (0.1 M, 2 mL), THF (20 mL). Yield: 0.58 g (80%).
1H NMR (500 MHz, D2O, pH 3.39) δH 3.42 (s, 1H, CH of triple
bond), 7.48-7.52 (m, 4H, aromatic); ring I δH 3.02-3.07 (dd, J )
8.5, 13.5 Hz, 1H, H-6), 3.18-3.22 (dd, J1 ) J2 ) 9.0 Hz, 1H,
H-4), 3.28-3.32 (m, 1H, H-2), 3.37-3.40 (dd, J ) 3.0, 13.5 Hz,
1H, H-6′), 3.85-3.88 (m, 1H, H-5), 3.96-3.99 (m, 1H, H-3),
6.04-6.05 (d, J ) 4.0 Hz, 1H, H-1); ring II δH 1.94-1.98 (ddd, J1
) J2 ) J3 ) 12.5 Hz, 1H, H-2ax), 2.37-2.41 (dt, J ) 4.0, 12.5
Hz, 1H, H-2eq), 3.28-3.32 (m, 1H, H-1), 3.47-3.50 (m, 1H, H-3),
3.67-3.71 (t, J ) 10.0 Hz, 1H, H-6), 3.96-3.99 (m, 1H, H-5),
4.16-4.20 (t, J ) 9.5 Hz, 1H, H-4); ring III δH 4.48-4.49 (dd, J
) 2.0, 4.0 Hz, 1H, H-2), 4.64-4.68 (m, 2H, H-3, H-4), 5.47 (s,

Fluoroquinolone-Aminoglycoside Hybrid Antibiotics
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8 2253
1H, H-1); ring IV δH 2.70-2.74 (dd, J ) 8.0, 13.5 Hz, 1H, H-6),
2.94-2.97 (dd, J ) 3.0, 13.5 Hz, 1H, H-6′), 3.55 (m, 1H, H-2),
3.65 (m, 1H, H-4), 4.11-4.15 (m, 2H, H-3, H-5), 5.21 (s, 1H, H-1).
13C NMR (125 MHz, D2O) δC 29.6 (C-2), 41.9 (C-6′), 42.3 (C-
6′′′), 50.2, 51.8, 52.5, 55.5, 68.6, 69.1, 69.5, 71.3, 72.4, 73.1, 74.1,
75.4, 76.4, 80.2, 80.3, 81.3 (CH of triple bond), 85.0, 86.3, 96.9
(C-1′, C-1′′′), 112.4 (C-1′′), 120.4, 122.7, 135.0, 138.8, 172.0 (CO).
MALDI TOF MS calcd for C31H49N7O13K ([M + K]+) m/e 766.3;
measured m/e 766.3.
(Perkin-Elmer). The concentration of half-maximal inhibition (IC50)
was obtained from concentration-response curves fitted to the data
of at least two independent experiments by using Grafit 5 software
(Leatherbarrow, R. J. Erithacus Software Ltd.: Horley, U.K., 2001).
DNA supercoiling activity was assayed with relaxed pBR322
DNA as a substrate (TopoGEN, Inc.) according to the manufac-
turer’s protocol. The standard reaction mixture (20 µL) contained
35 mM Tris-Cl, pH 7.5, 24 mM KCl, 4 mM MgCl2, 2 mM
dithiothreitol, 1.8 mM spermidine, 1 mM ATP, 6.5% glycerol, 0.1
mg/mL BSA, 12.5 ng/µL relaxed pBR322, and DNA gyrase protein.
The reaction mixture was incubated at 37 °C for 1 h and then was
terminated by addition of a loading dye and chloroform/isoamyl
alcohol (24:1) mixture. After a brief vortex, the blue aqua phase
was analyzed by electrophoresis in 1% agarose. One unit of
supercoiling activity was defined as the amount of DNA gyrase
required to supercoil 0.5 µg of plasmid in 1 h. The IC50 was defined
as the drug concentration that reduced the enzymatic activity
observed with drug-free controls by 50%.
DNA relaxation activity was assayed with supercoiled pBR322
DNA as a substrate (Inspiralis Ltd.) according to the manufacturer’s
protocol. The standard reaction mixture (20 µL) contained 40 mM
HEPES-KOH, pH 7.6, 100 mM potassium glutamate, 10 mM
Mg(OAc)2, 10 mM dithiothreitol, 1 mM ATP, 50 µg/mL albumin,
5 ng/µL relaxed pBR322, and TopoIV protein. The reaction mixture
was incubated at 37 °C for 30 min and then was terminated by
addition of 2 µL of 0.5 M EDTA, 3.5 µL of loading dye, and 20
µL of chloroform/isoamyl alcohol (24:1) mixture. After a brief
vortex, the blue aqua phase was analyzed by electrophoresis in 1%
agarose. One unit of relaxation activity was defined as the amount
of TopoIV required to relax 0.1 µg of plasmid in 30 min. The IC50
was defined as the drug concentration that reduced the enzymatic
activity observed with drug-free controls by 50%.
General Procedure for the Preparation of Hybrid Structures
(1a-q). A solution of compound 2 (0.06 mmol), compound 3 (0.05
mmol), [(CH3CN)4Cu]PF6 (0.025 mmol) in 7% solution of Et3N in
water (5 mL) was placed in a glass vial (25 mL). The vial was
closed with a stopper and heated in a domestic microwave oven
for 40 s at maximum power. Propagation of the reaction could be
monitored by TLC [CH2Cl2/MeOH/H2O/MeNH2 (33% solution in
EtOH), 10:15:6:15]. After completion, the mixture was purified on
a short column of Amberlite CG-50 (H+-form). The column was
sequentially washed by MeOH, MeOH/MeNH2 (33% solution in
EtOH) 95:5, MeOH/MeNH2 (33% solution in EtOH) 9:1, and
MeOH/MeNH2 (33% solution in EtOH) 4:1. Fractions containing
the product were combined, evaporated, redissolved in water, and
evaporated again to afford the free amine form of the product. The
product was dissolved in water, the pH was adjusted to 3.2 with
TFA (0.01 M), and the mixture was lyophilized to afford the TFA
salt of the final product, usually as a white foamy solid. Chemical
yields of the resulting hybrids 1a-q are given in Table 2, and their
complete analytical data are given in Supporting Information.
Antibacterial Activity. The MIC values were determined using
the double-microdilution method according to the National Com-
mittee for Clinical Laboratory Standards (NCCLS)44 with starting
concentration of 384 and 1.5 µg/mL of the tested compounds. All
the experiments were performed in duplicate, and analogous results
were obtained in two to four different experiments.
Resistance studies were performed in parallel with E. coli ATCC
35218 and B. subtilis ATCC 6633 strains in the presence of Cipro,
NeoB, Cipro/NeoB mixture (1:1 molar ratio), and the hybrid 1i.
After the initial MIC experiments, MICs were determined once in
2 days for 15 passages as follows: for each compound tested,
bacteria from the one-half MIC well were diluted 100-fold (50 µL
of the bacterial growth in a total of 5 mL of LB medium) and were
grown overnight at 37 °C. The OD600 of the bacteria was diluted
to yield 5 × 105 cells/mL in LB (according to a calibration curve)
and used again for MIC determination in the subsequent generation.
In parallel, MIC evolution during these subcultures was compared
concomitantly with each new generation, using bacteria harvested
from control wells (wells cultured without antimicrobial agent from
the previous generation). The relative MIC was calculated for each
experiment from the ratio of MIC obtained for a given subculture
to that obtained for first-time exposure.
Acknowledgment. The authors thank Prof. Albert M.
Berghuis (McGill University) for kindly providing us with the
plasmid pETSACG1, Prof. Shahriar Mobashery (University of
Notre Dame) for providing us with the plasmid pSF815 carrying
the AAC(6′)-APH(2′′) gene, Prof. S. B. Levy for kindly
furnishing us the clinical isolates of E. coli AG100A and
AG100B, and Dr. Ofer Reany for excellent technical assistance.
This work supported by research grants from the Israel Science
Foundation founded by the Israel Academy of Sciences and
Humanities (Grant No. 515/07) and by the Technion V.P.R. V.B.
acknowledges the financial support by the Center of Absorption
in Science, the Ministry of Immigration Absorption and the
Ministry of Science and Technology, Israel (Kamea Program).
Supporting Information Available: Analytical data, 1H and 13
C
NMR spectra, and HPLC chromatograms of purity determination
for all the hybrid structures (1a-q). This material is available free
of charge via the Internet at http://pubs.acs.org.
Biochemical Studies. The plasmid pETSACG1 carrying the
APH(3′)-IIIa gene (GenBank Accession No. V01547) was gener-
ously provided by Prof. A. Berghuis, McGill University. The
plasmid pSF815 carrying the AAC(6′)-APH(2′′) gene was gener-
ously provided by Prof. S. Mobashery, University of Notre Dame.
The plasmid pET9d carrying the APH(3′)-Ia gene was obtained
from New England Biolabs. Purification and kinetic analysis of
APH(3′)-IIIa were performed according to a previously described
procedure.19
References
(1) Magnet, S.; Blanchard, J. S. Molecular insights into aminoglycoside
action and resistance. Chem. ReV. 2005, 105, 477–498.
(2) Wright, G. D.; Berghuis, A. M.; Mobashery, S. Aminoglycoside
antibiotics. Structures, functions, and resistance. AdV. Exp. Med. Biol.
1998, 456, 27–69.
Protein translation inhibition by the different compounds was
quantified in a coupled transcription/translation assay by using E.
coli S30 extracts for circular DNA with the pBESTluc plasmid
(Promega), according to the manufacturer’s protocol. Translation
reactions (25 µL) that contained variable concentrations of the tested
compound were incubated at 37 °C for 60 min, cooled on ice for
5 min, and diluted with a dilution reagent (tris-phosphate buffer
(25 mM, pH 7.8), DTT (2 mM), 1,2-diaminocyclohexanetetraacetate
(2 mM), glycerol (10%), Triton X-100 (1%), and BSA (1 mg/mL))
into 96-well plates. The luminescence was measured immediately
after the addition of the luciferase assay reagent (50 µL; Promega),
and the light emission was recorded with a Victor3 plate reader
(3) Kaufhold, A.; Podbielski, A.; Horaud, T.; Ferrieri, P. Identical genes
confer high-level resistance to gentamicin upon Enterococcus faecalis,
Enterococcus faecium, and Streptococcus agalactiae. Antimicrob.
Agents Chemother. 1992, 36, 1215–1218.
(4) Udo, E. E.; Al-Sweih, N.; John, P.; Chugh, T. D. Antibiotic resistance
of enterococci isolated at a teaching hospital in Kuwait. Diagn.
Microbiol. Infect. Dis. 2002, 43, 233–238.
(5) Chandrakanth, R. K.; Raju, S.; Patil, S. A. Aminoglycoside-resistance
mechanisms in multidrug-resistant Staphylococcus aureus clinical
isolates. Curr. Microbiol. 2008, 56, 558–562.
(6) Ardic, N.; Sareyyupoglu, B.; Ozyurt, M.; Haznedaroglu, T.; Ilga, U.
Investigation of aminoglycoside modifying enzyme genes in methi-
cillin-resistant staphylococci. Microbiol. Res. 2006, 161, 49–54.

2254 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 8
PokroVskaya et al.
(7) Daigle, D. M.; Hughes, D. W.; Wright, G. D. Prodigious substrate
specificity of AAC(6′)-APH(2′′), an aminoglycoside antibiotic resis-
tance determinant in enterococci and staphylococci. Chem. Biol. 1999,
6, 99–110.
(8) Boehr, D. D.; Daigle, D. M.; Wright, G. D. Domain-domain
interactions in the aminoglycoside antibiotic resistance enzyme
AAC(6′)-APH(2′′). Biochemistry 2004, 43, 9846–9855.
(9) Kondo, S.; Hotta, K. Semisynthetic aminoglycoside antibiotics:
development and enzymatic modifications. J. Infect. Chemother. 1999,
5, 1–9.
(10) Ye, X.-S.; Zhang, L.-H. Aminoglycoside mimetics as small-molecule
drugs targeting RNA. Curr. Med. Chem. 2002, 929–939.
(11) Umezawa, H.; Hooper, I. R. Aminoglycoside Antibiotics; Springer-
Verlag: Heidelberg, Germany, 1982.
of Staphylococcus aureus. Bioorg. Med. Chem. Lett. 2003, 13, 2109–
2112.
(28) Yao, L.; Smith, B. T.; Aube, J. Base-promoted reactions of bridged
ketones and 1,3- and 1,4-haloalkyl azides: competitive alkylation vs
azidation reactions of ketone enolates. J. Org. Chem. 2004, 69, 1720–
1722.
(29) Alper, P. B.; Hendrix, M.; Sears, P.; Wong, C.-H. Probing the
specificity of aminoglycoside-ribosomal RNA interactions with
designed synthetic analogs. J. Am. Chem. Soc. 1998, 120, 1965–1978.
(30) Fridman, M.; Belakhov, V.; Yaron, S.; Baasov, T. A new class of
branched aminoglycosides: pseudo-pentasaccharide derivatives of
neomycin B. Org. Lett. 2003, 5, 3575–3578.
(31) van den Bos, L. J.; Codee, J. D.; van der Toorn, J. C.; Boltje, T. J.;
van Boom, J. H.; Overkleeft, H. S.; van der Marel, G. A. Thiogly-
curonides: synthesis and application in the assembly of acidic
oligosaccharides. Org. Lett. 2004, 6, 2165–2168.
(12) Vakulenko, S. B.; Mobashery, S. Versatility of aminoglycosides and
prospects for their future. Clin. Microbiol. ReV. 2003, 16, 430–450.
(13) Bremner, J. B.; Ambrus, J. I.; Samosorn, S. Dual action-based
approaches to antibacterial agents. Curr. Med. Chem. 2007, 14, 1459–
1477.
(32) Thomas, J. R.; Liu, X.; Hergenrother, P. J. Size-specific ligands for
RNA hairpin loops. J. Am. Chem. Soc. 2005, 127, 12434–12435.
(33) Okusu, H.; Ma, D.; Nikaido, H. AcrAB efflux pump plays a major
role in the antibiotic resistance phenotype of Escherichia coli multiple-
antibiotic-resistance (Mar) mutants. J. Bacteriol. 1996, 178, 306–308.
(34) Ida, T.; Okamoto, R.; Shimauchi, C.; Okubo, T.; Kuga, A.; Inoue, M.
Identification of aminoglycoside-modifying enzymes by susceptibility
testing: epidemiology of methicillin-resistant Staphylococcus aureus
in Japan. J. Clin. Microbiol. 2001, 39, 3115–3121.
(35) Schmitz, F. J.; Fluit, A. C.; Gondolf, M.; Beyrau, R.; Lindenlauf, E.;
Verhoef, J.; Heinz, H. P.; Jones, M. E. The prevalence of aminoglycoside
resistance and corresponding resistance genes in clinical isolates of
staphylococci from 19 European hospitals. J. Antimicrob. Chemother.
1999, 43, 253–259.
(36) Hainrichson, M.; Yaniv, O.; Cherniavsky, M.; Nudelman, I.; Shallom-
Shezifi, D.; Yaron, S.; Baasov, T. Overexpression and initial charac-
terization of the chromosomal aminoglycoside 3′-O-phosphotransferase
APH(3′)-IIb from Pseudomonas aeruginosa. Antimicrob. Agents
Chemother. 2007, 51, 774–776.
(37) McKay, G. A.; Thompson, P. R.; Wright, G. D. Broad spectrum
aminoglycoside phosphotransferase type III from Enterococcus: over-
expression, purification, and substrate specificity. Biochemistry 1994,
33, 6936–6944.
(38) Robicsek, A.; Strahilevitz, J.; Jacoby, G. A.; Macielag, M.; Abbanat,
D.; Park, C. H.; Bush, K.; Hooper, D. C. Fluoroquinolone-modifying
enzyme: a new adaptation of a common aminoglycoside acetyltrans-
ferase. Nat. Med. 2006, 12, 83–88.
(39) Vetting, M. W.; Park, C. H.; Hegde, S. S.; Jacoby, G. A.; Hooper,
D. C.; Blanchard, J. S. Mechanistic and structural analysis of
aminoglycoside N-acetyltransferase AAC(6′)-Ib and its bifunctional,
fluoroquinolone-active AAC(6′)-Ib-cr variant. Biochemistry 2008, 47,
9825–9835.
(40) Barnard, F. M.; Maxwell, A. Interaction between DNA gyrase and
quinolones: effects of alanine mutations at GyrA subunit residues
Ser(83) and Asp(87). Antimicrob. Agents Chemother. 2001, 45, 1994–
2000.
(14) Hubschwerlen, C.; Specklin, J. L.; Sigwalt, C.; Schroeder, S.; Locher,
H. H. Design, synthesis and biological evaluation of oxazolidinone-
quinolone hybrids. Bioorg. Med. Chem. 2003, 11, 2313–2319.
(15) Grapsas, I.; Lerner, S. A.; Mobashery, S. Conjoint molecules of
cephalosporins and aminoglycosides. Arch. Pharm. (Weinheim, Ger.)
2001, 334, 295–301.
(16) Johnson, D. M.; Jones, R. N. CQ-397 and CQ-414: antimicrobial
activity and spectrum of two fluoroquinolone-cephalosporin, dual-
action compounds with carboxamido bonds. Clin. Microbiol. Infect.
1997, 3, 335–344.
(17) Zhi, C.; Long, Z. Y.; Manikowski, A.; Comstock, J.; Xu, W. C.;
Brown, N. C.; Tarantino, P. M., Jr.; Holm, K. A.; Dix, E. J.; Wright,
G. E.; Barnes, M. H.; Butler, M. M.; Foster, K. A.; LaMarr, W. A.;
Bachand, B.; Bethell, R.; Cadilhac, C.; Charron, S.; Lamothe, S.;
Motorina, I.; Storer, R. Hybrid antibacterials. DNA polymerase-
topoisomerase inhibitors. J. Med. Chem. 2006, 49, 1455–1465.
(18) Hubschwerlen, C.; Specklin, J. L.; Baeschlin, D. K.; Borer, Y.; Haefeli,
S.; Sigwalt, C.; Schroeder, S.; Locher, H. H. Structure-activity
relationship in the oxazolidinone-quinolone hybrid series: influence
of the central spacer on the antibacterial activity and the mode of
action. Bioorg. Med. Chem. Lett. 2003, 13, 4229–4233.
(19) Hainrichson, M.; Pokrovskaya, V.; Shallom-Shezifi, D.; Fridman, M.;
Belakhov, V.; Shachar, D.; Yaron, S.; Baasov, T. Branched aminogly-
cosides: biochemical studies and antibacterial activity of neomycin B
derivatives. Bioorg. Med. Chem. 2005, 13, 5797–5807.
(20) Zhang, J.; Chiang, F. I.; Wu, L.; Czyryca, P. G.; Li, D.; Chang, C. W.
Surprising alteration of antibacterial activity of 5′′-modified neomycin
against resistant bacteria. J. Med. Chem. 2008, 51, 7563–7573.
(21) Drlica, K.; Zhao, X. DNA gyrase, topoisomerase IV, and the
4-quinolones. Microbiol. Mol. Biol. ReV. 1997, 61, 377–392.
(22) Hawkey, P. M. Mechanisms of quinolone action and microbial
response. J. Antimicrob. Chemother. 2003, 51 (Suppl. 1), 29–35.
(23) Zimmer, C.; Triebel, H.; Thrum, H. Interaction of streptothricin and
related antibiotics with nucleic acids. Biochim. Biophys. Acta 1967,
145, 742–751.
(24) Ren, Y. G.; Martinez, J.; Kirsebom, L. A.; Virtanen, A. Inhibition of
Klenow DNA polymerase and poly(A)-specific ribonuclease by
aminoglycosides. RNA 2002, 8, 1393–1400.
(25) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A
stepwise Huisgen cycloaddition process: copper(I)-catalyzed regiose-
lective “ligation” of azides and terminal alkynes. Angew. Chem., Int.
Ed. 2002, 41, 2596–2599.
(41) Walsh, C. Where will new antibiotics come from? Nat. ReV. Microbiol.
2003, 1, 65–70.
(42) Radzishevsky, I. S.; Rotem, S.; Bourdetsky, D.; Navon-Venezia, S.;
Carmeli, Y.; Mor, A. Improved antimicrobial peptides based on acyl-
lysine oligomers. Nat. Biotechnol. 2007, 25, 657–659.
(43) Mor, A. Peptide-based antibiotics: a potential answer to raging
antimicrobial resistance. Drug DeV. Res. 2000, 50, 440–447.
(44) National Committee for Clinical Laboratory Standards. Performance
Standards for Antimicrobal Susceptibility Testing; Fifth Information
Supplement: Approved Standard M100-S5; NCCLS: Villanova, PA,
1994.
(26) Sriram, D.; Yogeeswari, P.; Basha, J. S.; Radha, D. R.; Nagaraja, V.
Synthesis and antimycobacterial evaluation of various 7-substituted
ciprofloxacin derivatives. Bioorg. Med. Chem. 2005, 13, 5774–5778.
(27) Kerns, R. J.; Rybak, M. J.; Kaatz, G. W.; Vaka, F.; Cha, R.; Grucz,
R. G.; Diwadkar, V. U. Structural features of piperazinyl-linked
ciprofloxacin dimers required for activity against drug-resistant strains
JM900028N