Covalently linked kanamycin – Ciprofloxacin hybrid antibiotics as a tool to fight bacterial resistance

Article abstract of DOI:10.1016/j.bmc.2017.02.068

To address the growing problem of antibiotic resistance, a set of 12 hybrid compounds that covalently link fluoroquinolone (ciprofloxacin) and aminoglycoside (kanamycin A) antibiotics were synthesized, and their activity was determined against both Gram-n

Full text of DOI:10.1016/j.bmc.2017.02.068

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Covalently linked kanamycin – ciprofloxacin hybrid antibiotics as a tool to fight
bacterial resistance
Michal Shavit, Varvara Pokrovskaya, Valery Belakhov, Timor Baasov
PII:
S0968-0896(17)30091-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.02.068
BMC 13621
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Bioorganic & Medicinal Chemistry
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Accepted Date:
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3 February 2017
Please cite this article as: Shavit, M., Pokrovskaya, V., Belakhov, V., Baasov, T., Covalently linked kanamycin –
ciprofloxacin hybrid antibiotics as a tool to fight bacterial resistance, Bioorganic & Medicinal Chemistry (2017),
doi: http://dx.doi.org/10.1016/j.bmc.2017.02.068
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antibiotics as a tool to fight bacterial resistance
Michal Shavit, Varvara Pokrovskaya, Valery Belakhov and Timor Baasov*
The Edith and Joseph Fischer enzyme inhibitors laboratory, Schulich Faculty of Chemistry, Technion – Israel
Institute of Technology, Haifa 3200003, Israel

Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Covalently linked kanamycin – ciprofloxacin hybrid antibiotics as a tool to fight
bacterial resistance
Michal Shavit, Varvara Pokrovskaya, Valery Belakhov and Timor Baasov*
The Edith and Joseph Fischer enzyme inhibitors laboratory, Schulich Faculty of Chemistry, Technion – Israel Institute of Technology, Haifa 3200003, Israel
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
To address the growing problem of antibiotic resistance, a set of 12 hybrid compounds that
covalently link fluoroquinolone (ciprofloxacin) and aminoglycoside (kanamycin A) antibiotics
were synthesized, and their activity was determined against both Gram-negative and Gram-
positive bacteria, including resistant strains. The hybrids were antagonistic relative to the
ciprofloxacin, but were substantially more potent than the parent kanamycin against Gram-
negative bacteria, and overcame most dominant resistance mechanisms to aminoglycosides.
Selected hybrids were 42-640 fold poorer inhibitors of bacterial protein synthesis than the parent
kanamycin, while they displayed similar inhibitory activity to that of ciprofloxacin against DNA
gyrase and topoisomerase IV enzymes. The hybrids showed significant delay of resistance
development in both E. coli and B. subtilis in comparison to that of component drugs alone or
their 1:1 mixture. More generally, the data suggest that an antagonistic combination of
aminoglycoside-fluoroquinolone hybrids can lead to new compounds that slowdown/prevent the
emergence of resistance.
Available online
Keywords:
Aminoglycosides
Fluoroquinolones
Hybrid antibiotics
Antibiotic resistance
Delay of resistance
2
009 Elsevier Ltd. All rights reserved.
1
.
Introduction
to individual drugs, and may even decrease the incidence of
resistance mutations development .
7
Since most of the currently known antibiotics target a single
essential process in pathogenic bacteria, the development of
resistance against these antibacterial substances by either
spontaneous mutations or by horizontal transfer of resistance
1
genes is rather fast. In an attempt to slow down the evolution of
resistance, one approach that has been used with some clinical
2
success is combination therapy ; a combination (cocktail) of two
or more different antibiotics employing distinct mechanisms of
action. Since the drugs act with different mechanisms, there is a
very low probability that any cell will simultaneously gain
3
,4
resistance to both drugs . However, the combination therapy
effects in vitro do not necessarily correlate to in vivo outcomes due
to the varied pharmacokinetic properties of the different drugs in
5
,6
the combination . Furthermore, this strategy cannot address the
problem of multiple drug resistance (MDR), strains exhibiting
resistance to both drugs in combination, and thus requires
employment of other families of drugs.
To address some of the limitations of combination therapy,
another intriguing approach named “hybrid antibiotics” has been
7
,8,9,10
developed
. The strategy is to chemically connect two drugs
Figure 1. Structures of ciprofloxacin, neomycin B, kanamycin A, Cipro-Neo
that target bacterial cells through different modes of action into a
single hybrid molecule. The covalent connection of two drugs can
make the pharmacokinetic properties of the hybrid molecule more
predictable, can improve its toxicity profile and can lead to
increased retention . Furthermore, rationally designed linkers
that connect two drugs may lead to better inhibition of both drug
targets, overcoming or mitigating existing resistance mechanisms
B hybrids and the designed KanA-Cipro hybrids.
With the motivation of these potential advantages, we recently
reported on hybrids that had been synthesized by linking two
commonly used antibiotics, ciprofloxacin (Cipro) that belongs to
the fluoroquinolone class of antibiotics, and neomycin B (NeoB)
1
1,12

a representative of the aminoglycoside (AG) class of antibiotics13
Fig. 1). The obtained Cipro-NeoB hybrids were active against a
wide range of wild-type Gram-negative and Gram-positive
bacteria, and were also able to overcome common resistance
mechanisms to AGs. Furthermore, tested Cipro-NeoB hybrids
have demonstrated a significant delay of resistance formation in
both Gram-negative (Escherichia coli) and Gram-positive
toxic than the parallel Cipro-NeoB hybrids. Thirdly, the clinical
AG amikacin, which is derived from KanA by installation of (S)-
4-amino-2-hydroxybutanoyl (AHB) moiety at N-1 position, is one
of the currently used AGs that has low toxicity (LD50=300 mg/kg)
(
1
5
and good activity against bacterial strains resistant to KanA . It is
suggested that the attached flexible AHB moiety in amikacin
interferes effectively with its binding to the AG inactivating
enzymes and prevents its acetylation, phosphorylation and
(Bacillus subtilis) bacteria in comparison to that of each
1
6
component drug separately or their 1:1 mixture.
adenylation . Furthermore, previous studies have shown that for
the 4,6-disubstituted 2-deoxystrepamine family of AGs (like
To understand the mechanism by which the Cipro-NeoB
hybrids modulate the evolution of resistance, one such hybrid, and
the combination of its component drugs were further investigated
in terms of phenotypic and genotypic evolution of resistance in E.
coli, by using integrated high-throughput resistance measurements
KanA) the best tolerance for structural variations was observed at
17–19
position N-1
. Based on these collective data, we anticipated
that attachment of KanA through the N-1 position to Cipro will
result in a new series of KanA-Cipro hybrids with potentially low
toxicity, good activity against AG resistant strains, and with good
potential to delay new resistance development.
1
4
and genomic sequencing . The observed data indicated that the
Cipro-NeoB hybrids delay resistance development mainly because
of its ability to evade resistance mediated by the multiple antibiotic
resistance (mar) operon that regulates efflux systems. The data
also demonstrated that the component drugs in the hybrid are
responsible for two different but complementary functions: The
Cipro moiety inhibits bacterial growth whereas the NeoB moiety,
being highly hydrophilic, diminishes the effectiveness of mar
activation. Since the antibacterial activity of these hybrids does not
rely on the NeoB moiety binding to the ribosome, it was
hypothesized that the NeoB component may be substituted with
chemically similar structures, in order to try and find a
compromise between permeability of the resulting hybrid and
evasion of the mar pathway.
We used three different alkyne derivatives of KanA
(compounds 2a-c) and six azido derivatives of Cipro (compounds
3a-f) that were straightforwardly coupled (via click reaction) using
microwave-assisted heating to yield a library of 12 different
KanA-Cipro hybrids 1a-l (Scheme 1). Importantly, the spacers X
(Table 1, the Cipro moiety) and Y (the KanA moiety) were similar
1
3
to our previously reported Cipro-NeoB hybrids and were selected
to vary both the length and chemical nature of the linkages
between the two drugs. Scheme 2 illustrates the synthesis of the
alkyne derivatives of KanA, compounds 2a-c. All these
compounds were synthesized from the common intermediate
a
3
(a) [(CH3CN)4Cu]PF6, 7% Et N in water, microwave 40 sec.
Scheme 1. Synthetic strategy for the assembly of KanA-Cipro Hybrids 1a-l.
derivative of KanA, compound 5, which was obtained in two
To test this hypothesis, we have designed, synthesized and
biologically evaluated new variants of the previously studied
Cipro-NeoB hybrid structures, in which the NeoB moiety is
replaced by another common AG antibiotic, kanamycin A (KanA).
We report that the resulted KanA-Cipro hybrids (1, Fig. 1) are
active against both Gram-negative and Gram-positive bacteria,
overcome the most prevalent types of resistance mechanisms
associated with AGs, and significantly delay resistance acquisition
in both Gram-negative and Gram-positive bacteria.
chemical steps from the commercial KanA according to the
20,21
previously published procedure . Briefly, commercial KanA
was first selectively protected with the benzyloxycarbonyl (Cbz)
protection at its two amino groups: N-6’ and N-3 positions to
afford compound 4. Treatment of 4 with ethyl trifluoroacetate (in
DMSO) afforded compound 5 in quantitative yield. Reaction with
propargyl bromide in the presence of K
2 3
CO gave the protected
alkyne derivative of KanA, compound 6. Two deprotection steps,
13
Table 1. Ciprofloxacin-Azido Derivatives 3a-f that were used in this study.
2
.
Results and Discussion
2
.1. Design and synthesis of KanA-Cipro hybrids
We chose KanA as a target AG molecule for the preparation of
new hybrids for the following reasons. Firstly, while KanA is
highly hydrophilic it significantly differs from NeoB (Fig. 1).
KanA consists of 3 rings and four amino groups, while NeoB has
Compound
X
4
rings and six amino groups. KanA belongs to the 4,6-
disubstituted 2-deoxystrepamine family of AGs while NeoB is the
representative of the 4,5-disubstituted 2-deoxystrepamine family
of AGs. Secondly, although KanA possesses similar antibacterial
activity to that of the previously used NeoB, KanA (LD50=280
mg/kg) is significantly less toxic than NeoB (LD50=24 mg/kg).
Therefore, the target KanA-Cipro hybrids are more likely to be less
3a
3b
3c
-(CH
-(CH
2
)
)
2
-
-
2
6
-
CH
-(CH
-CH -mC
-CH -pC
2
CH(OH)CH
-O-(CH
-CH
-CH
2
-
-
-
-
3
d
2
)
2
2
)
2
3
e
2
6
H
H
4
2
3
f
2
6
4
2

Scheme 2. Synthesis of KanA alkyne derivatives 2a-c.
removal of the trifluoroacetate ester with methyl amine followed
by Cbz deprotection in the presence of HBr in acetic acid, yielded
the alkyne derivative of KanA, compound 2a. In the synthesis of
the other two alkyne derivatives of KanA, compounds 2b and 2c,
the alkyne moiety is connected to the KanA moiety via an amide
linkage. Therefore, for the assembly of these compounds, the
intermediate compound 5 was directly coupled with either 4-
pentynoic acid or 5-hexynoic acid in the presence of 1-
hydroxybenzotriazole hydrate (HOBT) and dicyclohexyl
carbodiimide (DCC) to give the corresponding protected alkyne
derivatives of KanA, compounds 7a and 7b, respectively (Scheme
Staph. Epidermis (Gram-positive). Resistant strains included E.
coli AG100A and AG100B; these are kanamycin resistant
r
laboratory strains that harbor Kan transposon Tn903 (and also
2
2
upregulate the active efflux system) .
The MIC data in Table 2 shows that all the hybrids (1a-l)
exhibit significant antibacterial activity. The activity of the hybrids
in Gram-negative bacteria was improved in comparison to that of
KanA, while all the hybrids exhibited significantly reduced
activity compared to that of Cipro in both Gram-negative and
Gram-positive bacteria. The hybrids displayed up to 4 fold and
3
2fold activity improvement in comparison to KanA, against E.
2
2
). Finally, removal of the ester (MeNH , MeOH) and Cbz (HBr,
coli R477-100 and E. coli 25922, respectively. The activity
improvement was especially high against the resistant E. coli
AG100A (32-260 fold) and E. coli AG100B (8-32 fold) strains. In
contrast, most of the hybrids showed similar or lower activity than
KanA against the Gram-positive B. subtilis and Staph. Epidermis,
strains that are susceptible to AGs.
HOAc) protections afforded the alkyne derivatives 2b and 2c,
respectively.
The azide derivatives of Cipro, compounds 3a-f (Table 1) were
synthesized by direct coupling of the commercial Cipro with the
corresponding bromoazides/chloroazides as described previously
In general, we would like to note that the observed antibacterial
data in Table 2 of the KanA-Cipro hybrids 1a-l are very similar to
that previously reported by us with the parallel Cipro-NeoB
1
3
by us . Finally, the KanA-alkyne derivatives 2a-c were reacted
with selected Cipro-azide derivatives 3a-f under microwave
conditions (~40 seconds) in the presence of organic base (7% Et
in water) and Cu(I) catalyst to afford 12 new KanA-Cipro hybrids
a-l in 25-95% isolated yields (Scheme 1, Table 2). The structures
of 1a-l were confirmed by a combination of different analytical
3
N
13
hybrids , in the following ways: (1) their better performance
against Gram-negative versus Gram-positive bacterial strains
tested; (2) SAR in terms of the nature of the X and Y spacers used
to connect Cipro with the AG moiety; and (3) their especially high
improvement in activity against the AGs-resistant strains E. coli
AG100A and E. coli AG100B.
1
1
13
tools, including 2D H- C HMQC and HMBC, 2D COSY, and 1D
selective TOCSY experiments, along with mass spectral analysis.
2
.2. Antibacterial activity tests of the KanA-Cipro hybrids
The hybrids 1a-l, along with KanA and Cipro (that were used
Since the physio-chemical properties of the hybrids are very
different from those of their component drugs, one could argue that
the observed improved activity of the hybrids against AGs-
resistant strains E. coli AG100A and E. coli AG100B (Table 2)
could be the result of the improved cell permeability of the hybrids
versus that of the parent drugs.
as controls), were initially tested against a panel of AG susceptible
and AG resistant strains by measuring minimal inhibitory
concentration (MIC) values (Table 2). Susceptible strains included
E. coli R477-100 and 25922 (Gram-negative), and B. subtilis and

a
Table 2. Chemical yields and MIC values of the hybrids 1a-l against selected non-pathogenic bacterial strains
MIC (µg/mL)
E.coli
R477-
E.coli
25922
E.coli
AG100B
E.coli
AG100A
Bacillus
subtilis
Staph.
Epidermis
b
b
Yield
(%)
-
1
00
Compound
Cipro
KanA
X
-
-
Y
-
-
0.02
12
6
3
6
0.02
48
3
1.5
6
0.05
384
-
12
12
24
36
<0.005
96
-
0.37
0.28
1.5
0.02
1.5
1.5
1.5
3
0.094
1.5
6
6
3
-
1
1
1
a
b
c
-(CH
CH(OH)CH
2
)
2
-
-CH
-CH
-CH
2
-
55
77
75
35
45
-CH
-CH
2
2
-
-
2
-
-
2
-pC
-(CH
-mC
6
H
4
-CH
-
-CH
2
2
1
d
2
)
6
-CH
-CH
2
-
-
12
36
12
24
6
12
24
18
1
e
-CH
2
6
H
4
2
-
-
2
1.5
1
f
-CH
2
CH(OH)CH
2
-CO(CH
2
)
2
-
35
12
6
24
1.5
9
36
1
1
1
g
h
i
j
-(CH
-O-(CH
-mC -CH
-CH
-CH
2
)
2
-
-CO(CH
2
)
2
-
70
15
45
90
30
25
6
3
6
18
24
3
24
24
48
48
24
-
0.75
0.75
1.5
3
0.75
-
6
9
9
6
3
18
24
18
18
6
-(CH
2
)
2
2
)
2
-
-
-
-
-CO(CH
-CO(CH
-CO(CH
2
)
2
)
2
)
2
-
-
-
-
-
12
24
36
6
-CH
-CH
2
6
H
4
2
2
2
1
2
-pC
-pC
6
H
4
2
1
k
-CH
-CH
2
6
H
4
2
-CO(CH
-CO(CH
2
)
3
1
l
2
CH(OH)CH
2
-
2
)
3
12
6
1.5
24
a
The MIC values represent the results obtained in parallel experiments with two different starting concentrations of the tested compound (384 µg/ml and 1.5
b
µg/mL ). Kanamycin resistant E. coli strains expressing increased active efflux system.
Table 3: Antibacterial activities of the hybrids 1b-k against E. coli XL1 blue and BL21 background strains and their engineered variants expressing resistance
enzymes
MIC (µg/mL)
BL21
MIC (µg/mL)
MIC (µg/mL)
MIC (µg/mL)
XL1
BL21
pET11d
0.003
MIC
ratio
1
>32
BL21
pETSACG1
0.003
a
a
a
a
Compd
Cipro
KanA
BL21
0.003
12
psf815
0.003
<384
MIC ratio
MIC ratio
XL1 blue
0.006
3
Blue pET9d
0.006
MIC ratio
1
>32
1
>32
1
<384
<384
<384
>128
1
1
b
c
0.2
0.09
0.3
0.1
1.5
1
0.3
0.1
1.5
1
0.3
0.1
1.5
1
3
3
3
3
1
1
1
1
1
1
1
1
1
1
d
e
f
g
h
i
j
k
0.8
0.8
1.5
0.2
0.8
0.8
0.8
0.8
0.8
0.8
1.5
0.4
0.8
1.5
1.5
0.8
1
1
1
2
1
2
2
1
0.8
0.8
1.5
0.4
0.8
1.5
1.5
0.8
1
1
1
2
1
2
2
1
0.8
0.8
1.5
0.6
0.8
1.5
0.8
0.8
1
1
1
3
1
2
1
1
12
12
24
24
12
12
24
6
12
24
24
18
12
24
24
-
1
2
1
1
1
2
1
-
a
The MIC ratios were calculated by dividing the MIC values against resistant strain by that against background strain
To test this issue, selected hybrids (compounds 1b-k) were
(pET9d), derived by transformation of E. coli XL1 blue with the
pET9d plasmid that encodes for the APH(3′)-Ia resistance enzyme,
which catalyzes phosphorylation at the 3′-OH of both neomycin
and kanamycin families of aminoglycosides. These enzymes are
among the most prevalent modes of resistance found in
examined against six isogenic E. coli strains (Table 3): two
background strains (E. coli BL21 and E. coli XL1 blue) and four
strains with harbored resistance enzymes APH(3')-Ia, AAC(6')-
APH(2''), APH(3')-IIIa, and APH(3')-IIb. Among the resistant
strains used were the E. coli (pSF815), E. coli (pET11d) and E.
coli (pETSACG1); these are laboratory resistant strains derived by
transformation of E. coli BL21 (background strain) with the
pSF815, pET11d and pETSACG1 plasmids, respectively. The
pSF815 encodes for the bifunctional AAC(6′)/APH(2′′) resistance
2
3–25
aminoglycosides resistance strains
. The data in Table 3 show,
that while the MIC values of KanA dramatically rose in the case
of resistant strains, the activities of the hybrids were almost
identical against background and resistance-carrying strains.
These observations indicate that the enzymes modifying the
majority of AG antibiotics are ineffective in the case of the hybrid
molecules. These data also suggest that the reason for the observed
sensitivity of AG resistant strains, E. coli (pSF815), E. coli
(pET11d), E. coli (pETSACG1) and E. coli (pET9d), is the
enzyme, which catalyzes acetylation of the amino group at 6′-NH
2
and phosphorylation at the 2′′-OH. The pET11d and pETSACG1
plasmids encode for the APH(3')-IIb and APH(3′)-IIIa resistance
enzymes, respectively. The last resistant strain used was E. coli

reduced activity of the resistance enzymes against the 1b-k hybrids
rather than the improved cell permeability of these hybrids.
KanA and the cocktail of Cipro/KanA (1:1 molar ratio) during 15
successive subcultures. The observed data are shown in Figure 3 as
a ratio of the measured MIC values after the 15 and first passages.
th
Table 4: Activity of Selected Hybrids as Inhibitors of DNA Gyrase, TopoIV,
and Bacterial Protein Synthesis.
The individual components of the hybrids show a high tendency
towards resistance development: the relative MIC values are ~30-
fold for Cipro and over 15-fold for KanA in both E. coli and in B.
subtilis. A relatively high tendency towards resistance development
was also seen for the cocktail Cipro + KanA (1:1 molar ratio): in
the E.coli the level of resistance development was in between the
levels of Cipro and KanA, while in B. subtilis it was somewhat
lower than that of both. The hybrids 1b and 1c, however, showed
very low propensity to resistance development in both bacterial
strains with a ratio of 1.33 and 2.0 respectively for 1b, and for 1c
the ratio was 4 in both strains under the same experimental
IC50 (M)
Compou
nd
a
b
c
DNA gyrase
1.3 ± 0.1
TopoIV
Protein synthesis
inactive
Cipro
8.6 ± 0.3
KanA
inactive
inactive
0.03 ± 0.01
8.14 ± 1.04
1.27 ± 0.25
10.69 ± 1.55
6.46 ± 0.50
19.18 ± 1.64
2.19 ± 0.33
1
a
-
-
1
b
5.9 ± 0.4
19.8 ± 0.6
1
c
2.6 ± 0.3
7.9 ± 0.4
13
conditions. We note that earlier studies from our and other
3
1,30
1d
1g
1k
-
-
>100
-
laboratories
have reported the similar, high level resistance
development under same experimental conditions for Cipro and for
AGs. To the best of our knowledge, however, the KanA-Cipro
hybrids studied here, and the Cipro-NeoB hybrids reported recently
9.9 ± 0.4
-
1
3,14
by us
are the first AG-fluoroquinolone hybrids for which the
a
Supercoiling assay with E. coli DNA gyrase. The IC50 was defined as the drug
delay of resistance development has been demonstrated7,28,32.
concentration that reduced the enzymatic activity observed with drug-free
controls by 50%. See experimental section for assay details.
b
2.4. Summary and conclusions
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.
In vitro transcription/translation assay with E. coli S30 extract system. See
The present study was designed to further scrutinize the concept
of hybrid drugs especially in regards to their ability to modulate the
evolution of resistance. Indeed, previous reports by Kishony and co-
workers have demonstrated that while the “synergistic” antibacterial
drug combinations can actually enhance the development of
resistance, “antagonistic” drug combinations have significantly
c
experimental section for assay details.
2
.3. Biological evaluation of the KanA-Cipro hybrids.
The selected hybrids were further tested for their potential to
function with a dual mode of action. For this purpose, the ability of
3
3,34
slowed the evolution of resistance . Such a benefit of antagonistic
drug combination in regards to the hybrid antibiotic was first
demonstrated by the synthesis and evaluation of Cipro-NeoB
the hybrids 1a-d, 1g and 1k to inhibit protein synthesis was
1
3
examined by using an in vitro transcription/translation assay . In
parallel, the hybrids 1b-c and 1g were also tested for the inhibition
of the enzymes that are targeted by the quinolones, DNA gyrase
1
3,7
hybrids ; the hybrids were antagonistic relative to Cipro and showed
significant delay of resistance evolution in both Gram-negative (E.
coli) and Gram-positive (B. subtilis) bacteria. However, it remained
unclear whether or not the ability of these hybrid drugs to delay the
development of resistance was a general phenomenon for the
1
3,26,27
and TopoIV
. The observed data in Table 4 show that the
tested hybrids are 42-640 times poorer inhibitors of bacterial
protein synthesis than the parent KanA, suggesting that their
interaction with the bacterial ribosome is significantly reduced.
However, the tested hybrids 1a-d, 1g and 1k displayed better
antibacterial activity compared to that of KanA (Table 2), likely
due to the antibacterial activity that is mediated through the Cipro
moiety rather than the KanA moiety. Furthermore, all three hybrids
displayed similar activities to that of Cipro in both the DNA gyrase
and TopoIV assays, except for 1g that did not show activity on the
TopoIV target, lending further support to this hypothesis. The IC50
3
5
aminoglycoside-fluoroquinolone hybrids.
These accumulative data for the KanA-Cipro hybrids correlate
very well with the mechanism by which the Cipro-NeoB hybrids
1
4
limit the evolution of resistance , and support the notion that the
antagonistic mechanism of overcoming resistance is a general
phenomenon for aminoglycoside-fluoroquinoline hybrids. We
anticipate that this approach will be beneficial for future drug
design of hybrid molecules intended to slowdown/prevent the
values measured here for Cipro against both the DNA gyrase and
3
6
TopoIV, are very similar to those previously reported26,17,27.
emergence of multidrug resistance not only in infectious diseases
7
,28
but also in cancer
.
Finally, the apparent contradiction between the observed inferior
antibacterial activity of the tested hybrids 1b-c and 1g compared
with Cipro (Table 2), and their similar activity against both the
DNA gyrase and TopoIV targets (Table 4), can be explained by the
reduced cell penetration of the hybrid structures in comparison to
Cipro. Indeed, the bigger size and net charge of the hybrids
compared to Cipro, could contribute to their reduced cellular
uptake.
3
.
Experimental section
3
.1. General Methods.
1H NMR spectra (including DEPT, 2D-COSY, 2D TOCSY,
1
D TOCSY, HMQC, HMBC) were routinely recorded on a Bruker
TM
Avance 500 spectrometer or 400 spectrometer, and chemical shifts
reported (in ppm) are relative to internal Me
as the solvent, and to HOD (δ =4.63) with D
NMR spectra were recorded on a Bruker Avance 500 spectrometer
or at 125.8 MHz, and the chemical shifts reported (in ppm) relative to
4
Si (δ =0.0) with CDCl
3
1
3
The ability to slow down the emergence of resistance is
probably one of the most important advantages of the hybrid
2
O as the solvent.
C
TM
7
,28,29
drugs
. To evaluate the potential of Cipro-KanA hybrids to
delay resistance development, we used a well-established
the residual solvent signal for CDCl
3
(δ =77.00), or to external sodium
O as the solvent.
3
0,31
procedure of selective pressure , which was successfully
2
,2-dimethyl-2-silapentane sulfonate (δ =0.0) for D
2
1
3
implemented by us . Briefly, two different bacterial strains, one
Gram-negative (E. coli ATCC 35218) and one Gram-positive (B.
subtilis ATCC 6633), were exposed to sub-inhibitory (1/2 MIC)
amounts of the tested hybrids 1b and 1c, along with the Cipro,
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 monitored by
TLC on Silica Gel 60 F254 (0.25 mm, Merck), and spots

Figure 2. Representative comparative data for the inhibition of DNA gyrase (panels A and B) and TopoIV (panels C and D) with Cipro and compound 1b.
A) A 1% agarose gel shows the inhibitory activity of 1b against DNA gyrase. Lane 1, relaxed DNA; lane 2, supercoiling reaction by DNA gyrase without
(
presence of inhibitor; lanes 3-8 are the same as lane 1 but in the presence of 1, 2.5, 5, 11, 33, and 100 µM of inhibitor 1b. (B) Semilogarithmic plot of in
vitro DNA gyrase supercoiling reaction inhibition, measured for Cipro and 1b. (C) A 1% agarose gel shows the inhibitory activity of 1b against TopoIV.
Lane 1, supercoiled DNA; lane 2, relaxation reaction by TopoIV without the presence of inhibitor; lanes 3-8 are the same as lane 1 but in the presence of
1
, 2.5, 5, 11, 33, and 100 µM of 1b. (D) Semilogarithmic plot of TopoIV inhibition, measured for Cipro and 1b. The percentages of the supercoiled DNA
were calculated from the electrophoresis images by using GelAnelyzer program, and plotted as functions of drug concentration. Each data point represents
the average of 2-3 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, KanA, Cipro+KanA mixture
(1:1 molar ratio), and hybrid structures 1b and 1c. Relative MIC in the normalized ratio of MIC obtained for a given subculture to MIC obtained upon first exposure.
were visualized by charring with a yellow solution containing
bond), 4.96-5.10 (m, 4H, CH
aromatic); ring I δ 3.37-3.54 (m, 5H, H-2, H-3, H-4, H-6, H-6'),
3.63-4.08 (m, 1H, H-5), 4.96-5.10 (m, 1H, H-1); ring II δ 1.29-
1.36 (ddd, J =J =J =12.5 Hz, 1H, H-2ax), 2.23-2.27 (dt, J=4.0,
12.5 Hz, 1H, H-2eq), 2.96-2.98 (m, 1H, H-1), 3.37-3.54 (m, 1H,
H-5), 3.63-4.08 (m, 3H, H-3, H-4, H-6); ring III δ 3.22-3.26 (dd,
2
of Cbz), 7.23-7.35 (m, 10H,
.
(
NH
4
)Mo
7
O24 4H
2
O (120 g) and (NH
4
)
2
Ce(NO
3
)
6
(5 g) in 10%
H
H
2
SO
4
(800 mL). All reactions were carried out under an argon
H
atmosphere with anhydrous solvents, unless otherwise noted.
Microwave assisted reactions were carried out in domestic
microwave oven Sauter SG251. Compound 5 (Scheme 2) was
prepared from commercially available KanA (Shanghai FWD
Chemicals Limited) according to previously published
1
2
3
H
J=4.0, 13.0 Hz, 1H, H-6), 3.31-3.34 (m, 1H, H-6'), 3.37-3.54 (m,
2H, H-2, H-4), 3.63-4.08 (m, 2H, H-3, H-5), 4.96-5.10 (m, 1H, H-
2
0,21
13
procedure . All chemicals unless otherwise stated, were
obtained from commercial sources.
1); C NMR (125 MHz, CD
3
OD/CDCl
3
) δ
C
31.2 (C-2), 35.0 (C-
6''), 40.9 (C-6'), 50.1, 55.7, 61.2 (triple bond), 66.7, 66.8, 67.7,
69.7, 70.4, 71.4, 72.5, 72.8, 73.4, 73.9, 75.2, 84.0, 85.5, 100.41 (C-
1'), 101.3 (C-1"), 114.87, 117.2, 127.8, 127.9, 128.0, 128.1, 128.4,
3
.2. Synthesis of Kanamycin-alkyne derivatives 2a-c.
Compound 5 (100 mg, 0.115 mmol) was dissolved in dry DMF
(
5 mL) and to the resulting solution were added potassium
carbonate (19 mg, 0.140 mmol) and propargyl bromide (11.3 µL,
.126 mmol). Reaction progress was monitored by TLC
MeOH/DCM 1:5), which indicated completion after overnight
136.1, 136.2, 156.5, 157.9, 158.9, 159.2. MALDI TOFMS calcd
+
for C39
H
49
F
3
N
4
O19Na ([M+Na] ) m/e 909.3; measured m/e 909.3.
0
(
Compound 2a. Compound 6 (100 mg, 0.113 mmol) was dissolved
in 33% solution of MeNH in EtOH (10 mL) and the resulting
2
stirring. The crude mixture was then purified by flash
solution was stirred at room temperature for 12 h. The reagent and
the solvent were removed by evaporation and the residue was
dissolved in AcOH (2 mL) and stirred at 15°C for 10 minutes after
which 30% solution of HBr in AcOH (0.5 mL) was added.
chromatography (silica, MeOH/DCM) to yield compound 6 (72
1
mg, 70%). H NMR (500 MHz, CD
3
OD/CDCl
3
) δ
H
2.44-2.45 (t,
-triple
J=2.5 Hz, 1H, CH of triple bond), 3.63-4.08 (m, 2H, -CH
2

Reaction
progress
was
monitored
by
TLC
2 2 2 2
monitored by TLC [CH Cl /MeOH/H O/MeNH (33% solution in
(
1
CH
2
Cl
2
/MeOH/H
2
O/MeNH
2
(33% solution in EtOH)
EtOH) 10:15:6:15], which indicated completion after 2 hours. The
pH of the reaction was then adjusted to neutral with 1N NaOH.
The crude mixture was evaporated to dryness and then purified by
0:15:6:15), which indicated completion after 1 hour. 1N NaOH
solution was added to the reaction mixture until pH became
neutral, and then the crude mixture was purified on a short column
of Amberlite CG-50 (H -form). The column was sequentially
flash chromatography (silica, MeOH/MeNH
2
) to yield compound
O, pH=3.00) δ 3.00 (t,
1H, CH of triple bond), 3.94-3.98 (dd, J=2.5, 15.5 Hz, 1H, -CH
triple bond), 4.07-4.11 (dd, J=2.5, 15.5 Hz, 1H, -CH -triple bond);
ring I δ 3.03-3.08 (dd, J=9.0, 13.5 Hz, 1H, H-6), 3.24-3.27 (t,
+
1
2b (40 mg, 40%). H NMR (500 MHz, D
2
H
washed by: MeOH, MeOH/MeNH
MeOH/MeNH (33% solution in EtOH) 9:1 and MeOH/MeNH
33% solution in EtOH) 4:1. Fractions containing the product were
2
(33% solution in EtOH) 95:5,
2
-
2
2
2
(
H
combined, evaporated, re-dissolved in water and evaporated again
to afford the free amine form of the product (50 mg, 75%). This
product was then dissolved in water; the pH was adjusted to 7.5
J=9.5 Hz, 1H, H-4), 3.35-3.39 (dd, J=3.5, 9.0 Hz, 1H, H-6'), 3.59-
3.60 (m, 1H, H-2), 3.80-3.85 (m, 2H, H-3, H-5), 5.55-5.56 (d,
J=4.0 Hz, 1H, H-1); ring II δ 1.94-2.01 (ddd, J =J =J =12.5 Hz,
H 1 2 3
with 0.01 M H
a white foamy solid. H NMR (500 MHz, D
2
SO
4
and lyophilized to give the sulfate salt of 2a as
1H, H-2ax), 2.55-2.59 (dt, J=3.9, 12.5 Hz, 1H, H-2eq), 3.40-3.45
(m, 1H, H-3), 3.60-3.76 (m, 1H, H-5), 3.81-3.93 (m, 3H, H-1, H-
1
2
O, pH=3.17) δ
H
3.00
(t, 1H, CH of triple bond), 3.94-3.98 (dd, J=2.5, 15.5 Hz, 1H, -
4, H-6); ring III δ
H
3.40-3.45 (m, 1H, H-3), 3.55-3.58 (dd, J=4.0,
CH
2
-triple bond), 4.07-4.11 (dd, J=2.5, 15.5 Hz, 1H, -CH
3.03-3.08 (dd, J=9.0, 13.5 Hz, 1H, H-6), 3.24-3.28
t, J=9.5 Hz, 1H, H-4), 3.35-3.38 (dd, J=3.5, 9.0 Hz, 1H, H-6'),
.60-3.76 (m, 1H, H-2), 3.81-3.93 (m, 2H, H-3, H-5), 5.55-5.56
d, J=4.0 Hz, 1H, H-1); ring II δ 1.94-2.01 (ddd, J =J =J =12.5
Hz, 1H, H-2ax), 2.56-2.58 (dt, J=4.0, 12.5 Hz, 1H, H-2eq), 3.40-
2
-triple
10.5 Hz, 1H, H-4), 3.60-3.76 (m, 2H, H-6, H-6'), 3.81-3.93 (m,
1
3
bond); ring I δ
(
3
(
H
2H, H-2, H-5), 5.10-5.11 (d, J=3.0 Hz, 1H, H-1). C NMR (125
MHz, D O) δ 25.4 (C-2), 35.9 (-CH -triple bond), 40.4 (C-6'),
2
C
2
47.7, 49.8, 50.0, 54.9, 55.6, 59.8 (C-6''), 65.2, 68.1, 68.6, 70.7,
H
1
2
3
71.8, 72.4, 73.0, 73.7, 76.9, 78.8, 83.0, 96.0 (C-1'), 100.4 (C-1").
+
MALDI TOFMS calcd for C21
H
39
N
4
O
11 ([M+H] ) m/e 587.3;
3
1
.45 (m, 1H, H-3), 3.60-3.76 (m, 1H, H-5), 3.81-3.93 (m, 3H, H-
, H-4, H-6); ring III δ 3.40-3.45 (m, 1H, H-3), 3.55-3.58 (dd,
measured m/e 587.3.
H
J=4.0, 10.5 Hz, 1H, H-4), 3.60-3.76 (m, 2H, H-6, H-6'), 3.81-3.93
Compound 2c. Compound 5 (200 mg, 0.23 mmol) was dissolved
in dry Et N (2 mL) and cooled to -10 C. The HOBT (217.42 mg,
3
1.61 mmol), 5-hexynoic acid (67.6 µL, 0.69 mmol) and DDC
1
3
o
(
(
6
7
m, 2H, H-2, H-5), 5.10-5.11 (d, J=3.0 Hz, 1H, H-1). C NMR
125 MHz, D O) δ 25.4 (C-2), 35.9 (-CH -triple bond), 40.4 (C-
'), 47.7, 50.0, 54.9, 55.6, 59.8 (C-6''), 65.2, 68.1, 68.6, 70.7, 71.8,
2.4, 73.0, 73.7, 76.9, 78.8, 83.0, 96.0 (C-1'), 100.4 (C-1").
2
C
2
(332.22mg 1.61mmol) were dissolved in DMF (5 mL), and stirred
o
o
for 1hr at 0 C. The two solutions were combined at -10 C, and the
resulting mixture was stirred for about 2 hours. The reaction
progress was monitored by TLC (MeOH/DCM 1:4), which
+
39 4
MALDI TOFMS calcd for C21H N O11 ([M+H] ) m/e 523.2;
measured m/e 523.3.
2
indicated completion after 2 hours. A solution of MeNH (20 ml,
Compound 2b. Compound 5 (200 mg, 0.23 mmol) was dissolved
33% methylamine in MeOH) was then added, and the resulting
solution was stirred overnight at room temperature. The crude
mixture was evaporated to dryness and thenpurified by flash
o
in dry Et
.61 mmol), 4-pentynoic acid (67.6 µL, 0.69 mmol) and DDC
332.22 mg 1.61 mmol) were dissolved in DMF (5 mL), and stirred
3
N (2 mL) and cooled to -10 C. The HOBT (217.42 mg,
1
(
chromatography (silica, MeOH/DCM) to yield compound 7b.
o
o
1
for 1hr at 0 C. The two solutions were combined at -10 C and the
reaction progress was monitored by TLC (MeOH/DCM 1:4)
(165 mg, 88%). H NMR (500 MHz, CD
3
OD/CDCl
3
) δ
-triple bond),
of Cbz), 7.25-7.35 (m, 10H, aromatic); ring
3.38-3.48 (m, 5H, H-2, H-3, H-4, H-6, H-6'), 3.63-3.81 (m,
1H, H-5), 4.96-5.10 (m, 1H, H-1); ring II δ 1.43-1.52 (ddd,
=J =J =9 Hz, 1H, H-2ax), 2.31-2.36 (dt, J=4.0, 12.5 Hz, 1H, H-
2eq), 2.83-2.86 (m, 1H, H-1), 3.37-3.54 (m, 1H, H-5), 3.63-4.08
(m, 3H, H-3, H-4, H-6); ring III δ 3.22-3.26 (dd, J=4.0, 13.0 Hz,
1H, H-6), 3.31-3.34 (m, 1H, H-6'), 3.37-3.54 (m, 2H, H-2, H-4),
H
2.61-2.73
(m, 1H, CH of triple bond), 3.63-4.08 (m, 2H, -CH
5.05-5.10 (m, 4H, CH
I δ
2
which indicated completion after 2 hours. A solution of MeNH
20 ml, 33% methylamine in MeOH) was then added, and the
2
2
(
H
resulting solution was stirred over night at room temperature. The
crude mixture was evaporated to dryness and then purified by flash
chromatography (silica, MeOH/DCM) to yield compound 7a (170
H
J
1
2
3
1
mg, 90%). H NMR (500 MHz, CD
3
OD/CDCl
3
) δ
H
2.48-2.55 (t,
-triple
of Cbz), 7.26-7.35 (m, 10H,
3.45-3.49 (m, 5H, H-2, H-3, H-4, H-6, H-6'),
.77-3.85 (m, 1H, H-5), 5.00-5.08 (m, 1H, H-1); ring II δ 0.88-
.92(ddd, J =J =J =12.5 Hz, 1H, H-2ax), 2.27-2.29 (dt, J=4.0,
2.5 Hz, 1H, H-2eq), 2.95-2.03 (m, 1H, H-1), 3.37-3.54 (m, 1H,
3.22-3.26 (dd,
J=4.0, 13.0 Hz, 1H, H-6), 3.31-3.34 (m, 1H, H-6'), 3.37-3.54 (m,
H
J=3.5 Hz, 1H, CH of triple bond), 3.34-3.35 (m, 4H, -CH
bond), 4.96-5.10 (m, 4H, CH
aromatic); ring I δ
2
1
3
2
3.63-4.08 (m, 2H, H-3, H-5), 4.96-5.10 (m, 1H, H-1); C NMR
(125 MHz, CD OD/CDCl ) δ 31.2 (C-2), 35.0 (C-6''), 40.9 (C-6'),
H
3
3
C
3
0
1
H
50.1, 55.7, 61.2 (triple bond), 63.4, 66.7, 66.8, 67.7, 69.7, 70.4,
71.4, 72.5, 72.8, 73.4, 73.9, 75.2, 84.0, 85.5, 100.41 (C-1'), 101.3
(C-1"), 114.87, 117.2, 127.8, 127.9, 128.0, 128.1, 128.4, 136.1,
1
2
3
H-5), 3.63-4.08 (m, 3H, H-3, H-4, H-6); ring III δ
H
136.2, 156.5, 157.9, 158.9, 159.2. MALDI TOFMS calcd for
+
C
42
H
53
F
3
N
4
O
17Na ([M+Na] ) m/e 951.3; measured m/e 951.4.
2
1
2
6
8
1
H, H-2, H-4), 3.63-4.08 (m, 2H, H-3, H-5), 4.96-5.10 (m, 1H, H-
1
3
); C NMR (125 MHz, CD
3
OD/CDCl
3
) δ
C
29.3, 29.5, 31.5 (C-
Compound 7b from the previous step (100 mg, 0.113 mmol)
was dissolved in 33% solution of MeNH in EtOH (10 mL) and
), 34.9 (C-6''), 41.1 (C-6'), 50.1, 53.6, 61.4 (triple bond), 66.5,
6.6, 67.7, 69.6, 70.5, 71.3, 72.5, 72.9, 73.4, 73.6, 75.4, 81.2, 84.0,
5.6, 100.6 (C-1'), 101.1 (C-1"), 114.9, 117.3, 127.8, 127.9, 128.0,
2
the resulting solution was stirred at room temperature for 12 h. The
reagent and the solvent were removed by evaporation and the
residue was dissolved in acetic acid (2 mL), stirred at 15°C for 10
minutes, after which 30% solution of HBr in AcOH (0.5 mL) was
added. Reaction progress was monitored by TLC
28.1, 128.4, 136.2, 136.4, 156.9, 157.5, 158.7, 159.0. MALDI
+
TOFMS calcd for C41
measured m/e 951.2.
H
51
F
3
N
4
O
17Na ([M+Na] ) m/e 951.3;
[
2 2 2 2
CH Cl /MeOH/H O/MeNH (33% solution in EtOH) 10:15:6:1],
Compound 7a from the previous step (100 mg, 0.113 mmol)
was dissolved in 33% solution of MeNH in EtOH (10 mL) and
which indicated completion after 2 hours. 1N NaOH solution was
added to the reaction mixture until pH became neutral. The crude
mixture was evaporated to dryness at room temperature and then
2
the mixture was stirred at room temperature for 12 h. The reagent
and the solvent were removed by evaporation and the residue was
purified by flash chromatography (silica, MeOH/MeNH
2
) to yield
O, pH=3.20)
H
δ 2.44-2.47 (t, J=5.0 1H, CH of triple bond), 3.91-3.96 (dd,
o
1
dissolved in acetic acid (10 mL), cooled to 16 C and the 30%
compound 2c (40 mg, 40%). H NMR (500 MHz, D
2
solution of HBr in AcOH (1 ml) was added. Reaction progress was

J=2.5, 15.5 Hz, 1H, -CH
Hz, 1H, -CH -triple bond); ring I δ
H, H-6), 2.54-2.67 (t, J=6.8 Hz, 1H, H-4), 3.34-3.37 (dd, J=3.5,
.0 Hz, 1H, H-6'), 3.60-3.76 (m, 1H, H-2), 3.81-3.93 (m, 2H, H-3,
1.75-1.86 (ddd,
=13.75 Hz, 1H, H-2ax), 2.32-2.35 (dt, J=4.0, 12.5 Hz, 1H,
H-2eq), 3.43-3.48 (m, 1H, H-3), 3.63-3.67 (m, 1H, H-5), 3.84-3.88
m, 3H, H-1, H-4, H-6); ring III δ 3.43-3.48 (m, 1H, H-3), 3.56-
.62 (dd, J=4.0, 10.5 Hz, 1H, H-4), 3.58-3.62 (m, 2H, H-6, H-6'),
2
-triple bond), 4.09-4.12 (dd, J=2.5, 15.5
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.
2
H
3.23-3.27 (dd, J=8.4, 12.9 Hz,
1
9
H-5), 5.55-5.56 (d, J=4.0 Hz, 1H, H-1); ring II δ
=J =J
H
J
1
2
3
3.5. Biochemical studies
Resistance conferring plasmids used in this study were
obtained as follows. The plasmid pSF815 carrying the AAC(6')-
APH(2'') gene was kindly provided by Prof. S. Mobashery,
University of Notre Dame. The plasmid pETSACG1 carrying the
APH(3')-IIIa gene (Gene bank Accession No. V01547) was
obtained from Prof. A. Berghuis, McGill University. The plasmid
pET9d carrying the APH(3')-Ia gene was from New England
Biolabs.
(
3
3
H
1
3
.81-3.93 (m, 2H, H-2, H-5), 5.22-5.23 (d, J=3.0 Hz, 1H, H-1). C
O) δ 17.6, 24.1 (C-2), 31.0, 35.2 (-CH -triple
bond), 40.2 (C-6'), 47.5, 50.5, 55.5, 60.1 (C-6''), 65.9, 68.0, 68.8,
NMR (125 MHz, D
2
C
2
7
1
0.8, 71.9, 72.8, 73.0, 73.9, 76.9, 79.2, 80.2, 84.8, 95.5, 98.0 (C-
'), 100.3, 115.1, 117.6, 133.0, 162.8, 176.2 (C-1"). MALDI
+
TOFMS calcd for C24
H
42
N
4
O
12 ([M+H] ) m/e 601.3; measured
DNA supercoiling and relaxation activities were assayed
m/e 601.1
according to the manufacturer s protocols by following the
1
5
procedures we described previously . Briefly, DNA supercoiling
activity was assayed with relaxed pBR322 DNA as a substrate
3
.3. Synthesis of KanA-Cipro hybrids 1a-l.
General procedure for the preparation of hybrid
(
TopoGEN, Inc) and the DNA relaxation activity was assayed with
supercoiled pBR322 DNA as a substrate (Inspiralis Ltd). The IC50
values in both experiments were defined as the drug concentration
that reduced the enzymatic activity observed with drug-free
controls by 50%.
DNA fragments were separated on 1% agarose gel using
TAEx1 buffer. Agarose was added to TAE buffer for a 1% (w/v)
concentration and heated with a microwave oven until fully
dissolved. Etidium Bromide was added to a slightly cooled
solution to a final concentration of 0.625μg/ml. Molecular weight
was determined with DNA standards λ DNA/HindIII (Fermentas).
structures 1a-l. This procedure was very similar to that previously
reported by us for the preparation of Cipro-NeoB hybrids (Fig.
1
3
1
) . Briefly, a solution of compound 2 (0.06 mmol), ciprofloxacin
1
3
azido derivative 3 (0.05 mmol) , and [(CH
3 4 6
CN) Cu]PF (0.025
mmol) in 7% solution of Et N in water (5 mL) was placed in a
3
glass vial (25 mL) compatible for working with microwave
irradiation. The vial was closed non-hermetically with a stopper
and carefully heated in a domestic microwave oven at maximum
power, in 5 second runs, for a total of 40 seconds. We note that the
volume of the solution (5 mL) and the 40 seconds of heating were
found to be optimal for the particular reactions reported here.
6
×Loading Dye (Fermentas) were added to each sample (1:1
ratio), loaded on to the gel and ran at 90mV in a Sub-Cell GT Cell
Bio-Rad) until separation (approximately 90min). DNA
Reaction
progress
was
monitored
by
TLC
(
[
CH
2
Cl
2
/MeOH/H
2
O/MeNH
2
(33% solution in EtOH),
fragments were examined under UV light (Uvitec).
1
0:15:6:15]. After completion, the mixture was purified on a short
Prokaryotic in-vitro translation inhibition was quantified in
+
column of Amberlite CG-50 (H -form). The column was
sequentially washed by: MeOH, MeOH/MeNH (33% solution in
EtOH) 95:5, MeOH/MeNH (33% solution in EtOH) 9:1 and
MeOH/MeNH (33% solution in EtOH) 4:1. Fractions containing
quick coupled transcription/translation assays by using E. coli S30
2
TM
extract for circular DNA with the pBESTluc
plasmid
2
(
Promega), according to the manufacturer’s protocol and the
2
13
detailed procedure described previously by us with some minor
modifications as follows. Translation reactions were performed in
a total volume of 10 µL (instead of 25 µL volume reported
previously) and the luminescence was measured immediately (into
the product were combined, evaporated, re-dissolved 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 lyophilized to afford the TFA salt of the final
product, usually as a white foamy solid. Chemical yields of the
resulting hybrids 1a-l are given in Table 2 and their complete
analytical data are given in Supporting Information.
9
6-well plates) after the addition of the luciferase assay reagent
(4.5 µL of reaction and 45µl dilution reagent; Promega). The IC50
values were obtained from fitting concentration-response curves
to the data of at least two independent experiments by using Grafit
5
software (Leatherbarrow, R. J. Erithacus Software Ltd.:
3
.4. Antibacterial activity
Horley, U.K., 2001).
For the determination of MIC values we used the double-
microdilution method according to the National Committee for
Clinical Laboratory Standards (NCCLS) with two different
Acknowledgments
3
7
starting concentrations of the tested compounds: 384 µg/mL and
The authors thank Prof. Shahriar Mobashery (University of
Notre Dame) for providing us with the plasmid pSF815 carrying
the AAC(6')-APH(2'') gene, Prof. Albert M. Berghuis (McGill
University) for kindly providing us with the plasmid pETSACG1,
and S. B. Levy for kindly providing us with the clinical isolates of
E. coli AG100A and AG100B. This work was supported by
research grants from the Israel Science Foundation founded by the
Israel Academy of Sciences and Humanities (grant no. 1845/14),
and from the Ministry of Science, Technology and Space, State of
Israel (grant no. 2022675). V.B. acknowledges the financial
support by the Ministry of Immigration Absorption and the
Ministry of Science and Technology, Israel (Kamea Program).
1
.5 µg/mL. All the experiments were performed as duplicates and
analogous results were obtained in two to four different
experiments. The evolution of resistance was studied in parallel
with E. coli ATCC 35218 and B. subtilis ATCC 6633 strains as we
1
3
reported earlier . Briefly, the experiments were performed in the
presence of Cipro, KanA, Cipro:KanA mixture (1:1 molar ratio),
and the hybrids 1b and 1c. MICs were determined for 15 passages
as follows: for each compound tested, bacteria from the 1/2 MIC
well were diluted 100-fold (50 µL of the bacterial growth in the
o
total of 5 mL LB medium) and were grown overnight at 37 C. The
5
OD600 of the bacteria was diluted to yield 5x10 cells/ml in LB
(determined by using a calibration curve) and used again for MIC
Supplementary Material
determination in the subsequent generation. Note that the MIC
evolution during these subcultures was compared concomitantly
with each new generation, using bacteria harvested from control
Supplementary data associated with this article can be found, in
the online version, at htpp://dx.doi.org/10.1016/j.bmc.2017……

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