Novel Glycoconjugate of 8-Fluoro Norfloxacin Derivatives as Gentamicin-resistant Staphylococcus aureus Inhibitors: Synthesis and Molecular Modelling Studies

Article abstract of DOI:10.1111/cbdd.12503

Antibiotic resistance has been the subject of interest in clinical practice due to high prevalence of antibiotic-resistant pathogenic organisms. In view of the prevalence of lesser resistance in antibiotics belonging to aminoglycoside class of compounds viz. Food and Drug Administration-approved gentamicin for the treatment of Staphylococcus infections, which also has instances of resistance in the clinical isolates of Staphylococcus aureus, a series of novel glycoconjugates of 8-fluoro norfloxacin analogues with high regio-selectivity by employing copper (I)-catalyzed 1, 3-dipolar cycloaddition of 1-O-propargyl monosaccharides has been synthesized and evaluated for the antibacterial activity against gentamicin resistance Staphylococcus aureus. Among these compounds, the compound 10g showed better antibacterial activity (MIC = 3.12 μg/ml) than gentamicin (Escherichia coli (12.5 μg/ml), Staphylococcus aureus (6.25 μg/ml) and Klebsiella pneumonia (6.25 μg/ml), including gentamicin resistant (>50 μg/ml) strain in vitro). The docking studies suggest DNA gyrase of Staphylococcus aureus as a probable target for the antibacterial action of compound 10g.

Full text of DOI:10.1111/cbdd.12503

Chem Biol Drug Des 2015; 86: 440–446
Research Article
Novel Glycoconjugate of 8-Fluoro Norfloxacin
Derivatives as Gentamicin-resistant Staphylococcus
aureus Inhibitors: Synthesis and Molecular Modelling
Studies
1
1,2
Chandra S. Azad , Shome S. Bhunia
,
of mRNA in the initial phase of protein synthesis, through
initiation of non-functional proteins in prone micro-organ-
isms (2). The action of gentamicin and other bactericides
inside the bacterial cell takes place in two phases by an
active transport mechanism. In the first phase, entrance
into the cell depends on the transmembrane potential pro-
duced by aerobic metabolism, while the previous union of
the aminoglycoside to the bacterial ribosome favours the
second phase. Certain conditions reduce the electrical
potential of the membrane, such as low pH or anaerobic
status of the medium and thus decreasing the accumula-
tion of these compounds into the bacterial cytoplasm (3).
3
3
Atul Krishna , Praveen K. Shukla and
1
,2,
Anil K. Saxena
*
1
Division of Medicinal and Process Chemistry, CSIR-
Central Drug Research Institute, Lucknow, UP 226031,
India
Academy of Scientific and Innovative Research, New
Delhi, India
Fermentation Technology Division, CSIR-Central Drug
Research Institute, Lucknow, UP 226031, India
Corresponding author: Anil K. Saxena, anilsak@gmail.com
2
3
*
Antibiotic resistance has been the subject of interest in
clinical practice due to high prevalence of antibiotic-
resistant pathogenic organisms. In view of the preva-
lence of lesser resistance in antibiotics belonging to
aminoglycoside class of compounds viz. Food and
Drug Administration-approved gentamicin for the treat-
ment of Staphylococcus infections, which also has
instances of resistance in the clinical isolates of Staph-
ylococcus aureus, a series of novel glycoconjugates of
Although GM has been effectively used against most bac-
terial infections including Staphylococcus spp., Klebsiella
pneumoniae, Escherichia coli, Serratia marcescens, Cit-
robacter spp., Enterobacteriaceae spp. and Pseudomonas
spp, nevertheless, its clinical use suffered a major limita-
tion due to the emergence of resistance in 1974 (4,5).
Resistance to gentamicin and the related aminoglycosides
amikacin, kanamycin, netilmicin, sisomicin and tobramycin
8
-fluoro norfloxacin analogues with high regio-selectiv-
0
is mediated by AAD(6 ) and APH(2″) activities catalyzed by
ity by employing copper (I)-catalyzed 1, 3-dipolar
cycloaddition of 1-O-propargyl monosaccharides has
been synthesized and evaluated for the antibacterial
activity against gentamicin resistance Staphylococcus
aureus. Among these compounds, the compound 10g
showed better antibacterial activity (MIC = 3.12 lg/ml)
than gentamicin (Escherichia coli (12.5 lg/ml), Staphy-
lococcus aureus (6.25 lg/ml) and Klebsiella pneumonia
a single bifunctional protein encoded by aacA-aphD (6).
This gene is carried by the IS256-flanked composite trans-
poson Tn4001, found on large staphylococcal multiresis-
tance plasmids such as pSK1 and pSK41, or on the
chromosome (7). There are three mechanisms of GM
resistance: (i) reduced uptake due to decreased cell per-
meability, (ii) alterations at the ribosomal binding sites and
(iii) production of aminoglycoside-modifying enzymes (8).
Some strains of Pseudomonas aeruginosa and other bac-
teria exhibit aminoglycoside resistance due to a transport
defect or membrane impermeabilization. This mechanism
is likely to be chromosomally mediated and results in
cross-reactivity to all aminoglycosides (9,10). The protein
channels termed as permeases which is found on the
bacterial cell wall is specific in the recognition and trans-
port of selective chemical substances including carbohy-
drates (11). The interactions between carbohydrates and
proteins have determining roles in many biological pro-
cesses including infection mechanisms, inflammation,
immunological processes, signal transduction and cell
differentiation. Most of the biogenic proteins are also
glycosylated, and the glycosyl part of these proteins
(6.25 lg/ml), including gentamicin resistant (>50 lg/ml)
strain in vitro). The docking studies suggest DNA gyr-
ase of Staphylococcus aureus as a probable target for
the antibacterial action of compound 10g.
Key words: drug discovery, molecular modeling, molecular
recognition, therapeutic target
Received 26 August 2014, revised 9 December 2014 and
accepted for publication 12 December 2014
Gentamicin (GM) is an aminoglycoside antibiotic used in
the treatment of several Gram-positive and Gram-negative
bacterial infections (1). Gentamicin acts by linking the ribo-
somal subunits 30S and 50S and blocking the translation
4
40
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12503

Gentamicin-resistant S. Aureus Inhibitors
influences their biological activity, although the molecular
basis for such processes is still uncertain (12–14). As the
cell membrane of bacteria recognizes the carbohydrates
during the transmembrane transport, hence, improvement
of cell permeability by incorporating carbohydrate moiety
to conventional small ligands may provide a rational to pre-
vent permeability-related resistance of aminoglycosides. In
this context, we have selected quinolone class of mole-
cules due to their inherent influence in the area of antimi-
crobial chemotherapy. These molecules potentially offer a
wide range of advantages including broad spectrum of
activity, combined with high potency, good bioavailability,
oral and intravenous formulations, high serum levels, large
volume of distribution representing ample concentration in
tissues and low incidence of side-effects (15,16). So we
have designed a prototype molecule (1) incorporating car-
bohydrate moieties on quinolone nucleus using molecular
hybridization approach and ‘click chemistry’. The applica-
tion of click chemistry has been explored in all aspects of
drug discovery, ranging from lead discovery through com-
binatorial chemistry and target-template in situ chemistry,
to proteomics and DNA research using bioconjugation
reactions (17). The usage of click chemistry in the discov-
ery of new medicinally important molecules delivers a
means for the fast exploration of chemical space, assisting
lead optimization by structure–activity relationship (SAR)
through the generation of analogue libraries (Figure 1)
Merck silica gel 60F-254 plates). The plates were visual-
ized first with UV illumination followed by charring with
2 4 3
10% H SO in CH OH. Flash column chromatography
was performed using silica gel (230–400 mesh). The sol-
vent compositions reported for all chromatographic sepa-
rations are on a volume/volume (v/v) basis. All glasswares
were dried in an open flame before use in connection with
an inert atmosphere. Solvents were evaporated under
reduced pressure. Tetramethylsilane (0.0 p.p.m.) was used
1
as an internal standard in
H NMR, and CDCl
3
13
(77.0 p.p.m.) was used in C NMR. The abbreviations
used to indicate the peak multiplicity were as follows: s,
singlet; bs, broad singlet; d, doublet; dd, double doublet;
t, triplet; q, quartet; m, multiplet; Hz, Hertz. FAB MS was
recorded on Jeol (Japan)/SX-102/DA 6000. Infrared spec-
trum was taken with KBr on Perkin-Elmer RX-1, Waltham,
MA, USA. Melting points were determined on a Buchi 535
digital melting point apparatus and were uncorrected. Ele-
mental analysis was performed on a Perkin-Elmer 2400 C,
H, N analyzer, and values were within ꢀ0.4% of the calcu-
lated values.
Bioevaluation methods
The susceptibility testing was performed by standard broth
micro-dilution method as per Clinical and laboratory
Standard Institute (CLSI)(20) guidelines using RPMI 1640
Medium buffered with MOPS [3-(N-morpholino) propane-
sulphonic acid] for fungal cultures and Mueller-Hinton Broth
(Difco, Franklin Lakes, NJ, USA) for bacterial cultures in 96-
well microtitre plates. The maximum concentration tested
was 50 lg/ml, and the inoculum load in each test well was
(18,19).
Several carbohydrate conjugates of 1-ethyl-6,8-difluoro-4-
oxo-7-(1H-1,2,3-triazol-1-yl)-1,4-dihydroquinoline-3-carbox-
ylic acid have been synthesized and biological evaluated
for their antibacterial activity in comparison with gentami-
cin. The antibacterial effect of quinolones is mediated
through inhibition of DNA gyrase; hence, we have per-
formed docking studies of our synthesized carbohydrate
conjugates with DNA gyrase to reveal important binding
interactions.
3
in the range of 1–5 9 10 cells. The plates were incubated
for 24–48 h for yeasts, 72–96 h for mycelial fungi at 35 °C
and 24 h for bacteria at 37 °C and read visually as well as
spectrophotometrically (Spectra max) at 492 nm for deter-
mination of minimal inhibitory concentrations (MICs).
Modelling studies
Experimental
Ligand and protein preparation
General chemistry
The protein preparation was performed using Protein Prepa-
ration Wizard (21) implemented in Schr o€ dinger 9 suite . The
a
Reagent grade solvents were used for the extraction and
flash chromatography. All the reagents and chemicals
were purchased from Sigma–Aldrich Chemical Co. (St
Louis, MO, USA), Lancaster, and were used directly with-
out further purification. The progress of reactions was
checked by analytical thin-layer chromatography (TLC,
3D structures of the ligands were sketched in the Maestro
workspace using the drawing tools in maestro window. The
3D structures were geometrically optimized by clean up
geometry, and subsequent ligand preparation was per-
b
formed utilizing the LIGPREP module in MAESTRO (version 9.0).
Molecular Docking and Scoring
The molecular docking of the compounds was executed
c
using the GLIDE XP (extra precision) module implemented
a
in Schr o€ dinger 9 suite . The receptor grid was generated
using the ligand ciprofloxacin present in the crystal struc-
c
ture with pdb id 2XCT . Visualization of the docked confor-
Figure 1: The structure of prototype molecule.
Chem Biol Drug Des 2015; 86: 440–446
mation was performed in PyMOL.
441

Azad et al.
Chemistry
Biological activity
The required key intermediate quinolone 6,7,8 trifluoro
quinolone-3–carboxylate (7) was synthesized according to
the reported method (22), starting from 1,2,3 trifluoro aniline
The evaluation of antibacterial activities of compounds
10a-h against various strains of the bacteria, for example,
E. coli (ATCC 9637), Pseudomonas aeruginosa (ATCC
BAA-427), Staphylococcus aureus (ATCC 25923),
Staphylococcus aureus (ATCC ATCC 700699 methicillin
resistant), Staphylococcus aureus (ATCC 29213), Staphy-
lococcus aureus (ATCC 33592 gentamicin resistant), Kle-
bsiella pneumoniae (ATCC 27736), was carried out
according to the broth micro-dilution technique described
by NCCLS. The minimum inhibitory concentration (MIC) of
each compound was determined against test isolates
using this technique. The MICs of standard antibacterial
drug gentamicin were determined using Muller-Hinton
broth.
(
2) and diethyl 2-(ethoxymethylene) malonate (EMME, 3),
which on condensation followed by the thermal cyclization
in diphenyl ether of the condensed product 4 thus formed to
2 3
give ester 5. The N alkylation of 5 with ethyl iodide by K CO
in DMF yielded the N-ethylated derivative 6. The substitution
of C-7 F in quinolone ester 6 by azide was carried out by
NaN (3 equivalent) in DMF at 120 °C (Figure 2).
3
The azide 7 was then subjected to hydrolysis in basic condi-
tion using 2N NaOH, which resulted in insoluble mass, so
the acidic hydrolysis by HCl was carried out which yielded
the acid 8 in excellent yield. The acid 8 was kept in airtight
container because it turns orange-red when left in open air.
The antibacterial activity was compared with gentamicin
which was used as positive control. In all tests, the MIC
values are expressed in lg/mL. The Compounds 10a,
10b, 10f and 10h (MIC 12.5 mg/mL) had same activity
when compared with gentamicin (MIC 12.50 lg/mL), while
the compound 10g (MIC 6.25 lg/mL) showed better anti-
bacterial activity than gentamicin (MIC 12.50 lg/mL)
against E. coli. The Compound 10g (MIC 3.25 lg/mL) also
exhibited excellent antibacterial activity than gentamicin
against Staphylococcus aureus resistant to GM (Table 1).
The Compound 10g (MIC 3.25 mg/mL) also exhibited anti-
bacterial activity than GM against Klebsiella pneumoniae
(Table 1).
After synthesizing the azide counter part of 1, the alkyne
part of the prototype molecule (1) was planned for
synthesis. The alkyne counterpart was synthesized from
commercially available carbohydrates (D-arabinose,
D-galactose, D-glucose, D-mannose, N-Ac-glucosamine,
D-ribose, D-xylose and L-rhamnose) in excess of propargyl
alcohol using silica-supported sulphuric acid (SiO -H SO )
2
2
4
as an acid catalyst (23). The glycosylated sugar was fur-
ther acetylated by the same sulphuric acid (SiO -H SO ) in
2
2
4
excess of acetic anhydride without further purification. This
glycosylation reaction completed with -selectivity in most
of the cases. The coupling reaction between azide and
alkyne was performed in tert-butanol water (1:1) using
copper sulphate and ascorbic acid as a reducing agent.
The reaction was monitored by TLC, and after completion
of the reaction, the compounds were isolated and purified
by the flash chromatography. These compounds were
characterized by their IR, NMR and mass spectra
Docking studies
Bacterial type IIA topoisomerases (DNA gyrase and topo-
isomerase IV (or topo IV)) are the targets of the quinolone
antibiotics that have been in practice since 1962 (24).
However, direct evidences regarding the interaction of
quinolones with DNA gyrase or DNA were available after
(
Figure 3).
Figure 2: Reagents and conditions: (i) neat,
1
C
10–120 °C, 2 h; (ii) Ph
I, K CO , DMF, 95 °C, 10 h; (iv) NaN
DMF, 90–95 °C, 8 h; (v) 2 (N) HCl, reflux,
h; (vi) CuSO .5H O, NaASc, t-ButOH:H
1:1).
2
O, 250 °C, 8 h; (iii)
2
H
5
2
3
3
,
8
(
4
2
2
O
4
42
Chem Biol Drug Des 2015; 86: 440–446

Gentamicin-resistant S. Aureus Inhibitors
Figure 3: Structure of synthesized
compounds.
Table 1: Antibacterial activity of the synthesized compounds
Minimum inhibitory concentration (MIC) in lg/ml against
BACTERIA
1
Compound
2
3
3a
3b
3c
4
1
1
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
12.5
12.5
>50
>50
50
12.5
6.25
12.5
12.5
>50
>50
>50
>50
>50
>50
>50
>50
6.25
6.25
>50
>50
12.5
6.25
3.12
12.5
6.25
>50
>50
>50
>50
>50
>50
>50
>50
>50
6.25
6.25
>50
>50
12.5
6.25
3.12
12.5
0.78
6.25
6.25
>50
>50
12.5
6.25
3.12
12.5
>50
6.25
6.25
>50
>50
25
6.25
3.12
25
6.25
0g
0h
Gentamicin
3.12
1
. E. coli (ATCC 9637), 2. Pseudomonas aeruginosa (ATCC BAA-427), 3. Staphylococcus aureus (ATCC 25923), 3a. Staphylococcus aur-
eus (ATCC 700699 methicillin resistant), 3b. Staphylococcus aureus (ATCC 29213), 3c. Staphylococcus aureus (ATCC 33592 gentamicin
resistant), 4. Klebsiella pneumoniae (ATCC 27736).
the discovery of crystal structure that provided insights
about inhibitory function of quinolones. To reveal the
important interactions of our synthesized compounds at
the target site, we have performed docking studies that
may suitably establish the structure activity relationship
between these compounds. The co-crystalized structure
of protein in complex with ciprofloxacin (pdb id 2XCT)
was used for molecular docking (25). The fluoroquinol-
ones such as ciprofloxacin and norfloxacin exert
their antibacterial effect by acting on DNA gyrase. The
Chem Biol Drug Des 2015; 86: 440–446
443

Azad et al.
synthesized compounds are sugar conjugates of norfloxa-
cin; hence, we have selected DNA gyrase as the target
for these molecules (26,27). The results of the docking
studies revealed that the most active compound 10g hav-
ing the norfloxacin moiety resided at the ciprofloxacin site
but in an inverted conformation with the acidic moiety
incorporated within DNA strands and the xylose portion
incorporated in DNA gyrase.
tion with cytosine base pair of DNA. Besides having
hydrogen bond contact with DNA base, the sugar por-
tion in 10g was found to maintain contact with GLU
1088, GLY 1117, ALA 1118 and ALA 1120 (site A) as
depicted in Figure 4A. The least active molecule 10c
has its norfloxacin moiety aligned in a similar conforma-
tion as found in 10c, but the conjugated glucose moiety
failed to attain similar contact with DNA gyrase like the
xylose sugar in 10g (Table 2). The crystal structure of
ciprofloxacin depicts electron density map near SER
1084, implicating it to be an important residue for bind-
ing (Figure 5).
The negative fluorine atom in 10g forms hydrogen bond
with SER 1084, and the oxygen atom in the ring of
xylose sugar interacted through hydrogen bond forma-
Table 2: The glide docking score for the most and least active compound
Compound
Glide score
Glide evdw
Glide ecoul
Glide energy
XP Hbond
1
0g
0c
ꢁ6.19
ꢁ3.26
ꢁ5.66
ꢁ41.490
ꢁ73.90
ꢁ30.45
ꢁ19.57
ꢁ4.21
ꢁ2.20
ꢁ61.08
ꢁ78.12
ꢁ32.65
ꢁ0.7
1
ꢁ0.67
ꢁ0.65
Ciprofloxacin
A
B
Figure 4: (A) The docked conformation of
the most active compound 10g; (B) the
docked conformation of the least active
compound 10c; (C) the aligned
conformation of the most and the least
active compound at the binding site; (D) the
aligned conformation of the most active
compound 10g and ciprofloxacin at the
binding site.
C
D
Figure 5: The 2D ligand protein interaction
for (A) most active compound 10g and (B)
least active compound 10c without DNA
with major interactions with DNA gyrase.
B
A
4
44
Chem Biol Drug Des 2015; 86: 440–446

Gentamicin-resistant S. Aureus Inhibitors
The fluorine atom in the norfloxacin moiety occupied a
position similar to the carboxylic moiety in ciprofloxacin
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1
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resistant S. aureus (MIC > 50 lg/ml) and are equipotent
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1
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Appendix S1. General information and Experimental
procedure.
Appendix S2. Materials and Methods for biological
evaluation.
1
13
Figure S1. HNMR and CNMR spectra of selected
61:377–392.
compounds.
4
46
Chem Biol Drug Des 2015; 86: 440–446