Design, synthesis and docking studies of a novel ciprofloxacin analogue as an antimicrobial agent

Article abstract of DOI:10.1155/2012/340451

The carboxylic acid group of ciprofloxacin was modified and amino mercapto triazole was substituted. The compound was confirmed by physical parameters (solubility, melting point), chromatographic methods (TLC) and consistent with its IR & 1HNMR spectra. The synthesized analogue was screened for antibacterial activity against one gram positive & two gram negative species. The compound exhibited good antibacterial effect towards gram negative species when compared to the standard ciprofloxacin. At the same time the analogue was retaining antibacterial activity towards gram positive species when compared to standard ciprofloxacin. The molecular docking studies showed a good correlation between their antibacterial activity and autodock binding free energy.

Full text of DOI:10.1155/2012/340451

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
http://www.ejchem.net
2012, 9(2), 980-987
Design, Synthesis and Docking Studies of a Novel
Ciprofloxacin Analogue as an Antimicrobial AGENT
*
S.JUBIE , R. KALIRAJAN and PAVANKUMAR YADAV,
Department of Pharmaceutical Chemistry
J.S.S. College of Pharmacy, off campus - JSS University, Mysore
jubiejawahar@gmail.com
Received 31 July 2011; Accepted 4 October 2011
Abstract: The carboxylic acid group of ciprofloxacin was modified and amino
mercapto triazole was substituted. The compound was confirmed by physical
parameters (solubility, melting point), chromatographic methods (TLC) and
1
consistent with its IR & HNMR spectra. The synthesized analogue was
screened for antibacterial activity against one gram positive & two gram
negative species. The compound exhibited good antibacterial effect towards
gram negative species when compared to the standard ciprofloxacin. At the
same time the analogue was retaining antibacterial activity towards gram
positive species when compared to standard ciprofloxacin. The molecular
docking studies showed a good correlation between their antibacterial activity
and autodock binding free energy.
Keywords: Ciprofloxacin, 1,2,4-Triazole, Molecular docking, Antimicrobial
Introduction
Quinolones have become a major class of antibacterial agents, which are under extensive
clinical development. They have an attraction because of their extremely potent activity,
1
rapid bactericidal effects and low incidence of resistance development . The main
disadvantage of the quinolones is their limited activity against gram positive pathogens and
2
methicillin-resistant Staphylococcus aureus (MRSA) . In addition, quinolones can cause
certain adverse effects, such as CNS effects, phototoxicity, tendonitis, hypoglycemia, and
3
-4
serious cardiac dysrhythmias . Thus, despite many advances in the fluoroquinolone field,
there exists continuous need for novel quinolones with better activity profile,
pharmacokinetics and tolerability, to overcome the limitations of existing drugs.
Ciprofloxacin is a second generation fluoroquinolone used to treat various bacterial
infections. It is more effective against gram- negative organisms than gram- positive ones.
This moderate activity against some of the gram positive species limited its use in bacterial
5
infections .
Molecular docking plays an important role in the rational design of drugs. In the field of
molecular modeling, docking is a method which predicts the preferred orientation of one
molecule to a second when bound to each other to form a stable complex. Molecular docking

Design, Synthesis and Docking Studies of a Novel Ciprofloxacin 981
can be defined as an optimization problem, which would describe the “best-fit” orientation of a
6
ligand that binds to a particular protein of interest . 1, 2, 4-Triazole was reported to have a
7
-8
9-11
broad spectrum of antibacterial activity . Therefore on continuation of our work , in this
study we introduced 1, 2, 4-triazole into the quinolone antibacterial ciprofloxacin at its C-3
position and screen their antibacterial activity. Molecular docking studies was then employed
for the analysis with training set composed synthesized compound whose inhibitory activity is
unknown, in order to find out the molecular facilities responsible for biological activities.
Experimental
Melting points were taken in glass capillary tubes on a Veego VMP-1 Apparatus and are
uncorrected. The synthetic work was done by using CATALYST Scientific microwave
1
synthesis system. The H-NMR were recorded on Bruker DRX-300 (300 MHz FT-NMR)
using DMSO as solvent and TMS as internal standard. The IR spectra of compounds were
recorded on Shimadzu FT-IR spectrometer using KBr pellet technique and are expressed in
-
1
1
cm . HNMR spectra were recorded on a DPX-300 MHz Bruker FTNMR spectrometer
Bruker, USA). Chemical shifts were reported as parts per million (∂ ppm) with tetramethyl
(
silane (TMS) as an internal standard.
Synthesis of thiocarbohydrazide (1)
Hydrazine hydrate (0.3 mol, 9 mL) and 2-chloro ethanol (0.015 mol, 0.5 mL) were added to three
neck flask. Carbon disulphide (0.1 mol, 3 mL) was added drop wise with stirring in ice bath, a
yellow solid is formed. Finally sodium hydroxide (0.015 mol, 0.3 g) was added and subjected to
microwave irradiation at 400W for 15 minutes. The reaction mixture was cooled and the solid
12
obtained by filtration was washed by methanol and re crystallized from boiling water .
-
1
TLC: Chloroform: Methanol (9:1) IR (KBr cm ): 3022.55 (NH str), 2918.40 (Ar C-H
2
str), 3211.59 (NH str), 1143.8 (C-N str), 680.89 (C=S str), 977.94(N-C-S str).
Synthesis of triazole substituted ciprofloxacin (2)
Ciprofloxacin (0.3314 g, 0.001 mol) and thiocarbohydrazide (0.212 g, 0.002 mol) are taken
in a round bottom flask .The reaction mixture was kept under reflux in a water bath for 12 h.
Then the reaction mixture was cooled. The product formed was filtered, dried and
crystallized from ethanol.
-
1
TLC: Chloroform: Methanol (9:1) IR (KBr cm ): 3265.59(NH str heterocyclic),
257.63(C-N str), 1192.05(SH str) 1720.56 (C=O str), 1012.10(C- Fstr), 1238.34(N-N-Cstr),
1
9
02.72(C=Sstr). NMR (ppm) : 8.6 (s, 1H, N-CH), 2.0 (S, 2H, NH ), 7.5 (m, 3H, Ar-H), 3.0
2
(
s, 1H, SH), 3.2 (m, 8H, piperazine), 1.2 (d, 5H, cyclopropyl).
Molecular docking studies
Protein structure retrieval
The PDB is a key isomer in areas of structural biology, is a key repository for 3D structure
data of large molecule. The protein was extracted from protein database at the NCBI.
Lead preparation and energy minimization
The synthesized compounds which are derivatives of Ofloxacin were taken for prediction of 3D
structures by using Cambridge software. The energy was minimized for flexible docking using
Argus lab. The structures of these synthesized compounds and enzyme are shown in Figure 1.

9
82 S. JUBIE et al.
CFa
CFb
CFc
CFd
CFe
CFf
CFg
CFh
CFi
Figure 1. Different modes of ciprofloxacin.

Design, Synthesis and Docking Studies of a Novel Ciprofloxacin 983
2a
2b
2c
2d
2e
2f
2g
2h
Figure 2. Continued.

9
84 S. JUBIE et al.
2i
Figure 2. Different modes of synthesized compounds.
Receptor grid generation (auto dock vina 4.0)
In the receptor Grid Generation the receptor structure was defined by excluding any co-
crystallized ligand that may be present, determine the position and size of the active site as it
will be represented by receptor grids.
Molecular docking
During the docking process, initially Auto dock Vina performs a complete systematic search
of the conformational, oriental and positional space of the docked ligand and eliminating
unwanted conformations using scoring and followed by energy optimization. Predicting the
binding affinity and rank-ordering ligands in database screens was implemented by modified
and expanded version of the Chemscore 18 scoring function, for use in. For our studies,
x-ray crystal structure of synthesized compounds was taken from PDB, having resolution of
0
0
.375 A .Solvent molecules were deleted and bond order for crystal ligand and protein were
adjusted and minimized. Using standard precision (SP) mode of PyMOL software, docking
13
studies was performed on synthesized compounds .
Antimicrobial evaluation
All the synthesized compounds were screened for their antibacterial activity against
1
4
B.subtilis, K.pneumoniae & P.aeruginosa by using disc diffusion method . Bacteria were
cultured in nutrient agar medium and used as inoculum for study. The test compounds were
dissolved in N, Ndimethyl formamide (DMF) to obtain a solution of 25, 50 and100 μg/mL
concentration. The data are given in Table 1. The compounds were also screened for their
in-vitro antifungal activities against A. Niger, C.albicans & R.nigricans. The inhibitory
activities were compared with the commercial fungicide griseofulvin.The compounds
exhibited varying degree of activity. The data are given in Table 2.
Table 1. Different docking modes of ciprofloxacin&triazole substituted ciprofloxacin.
B.subtilis
Conc, µg/mL
50,
K.pneumoniae
Conc, µg/mL
50,
P.aeruginosa
Conc, µg/mL
50, 100,
Com
2
5,
100,
25,
100,
25,
Con µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/mL µg/ mL µg/mL
±
1
S.E
2.3
±S.E
13.6
±S.E
17.3
±S.E
11
±S.E
±S.E
17.6
±S.E
11.6
±S.E
13.3
±0.33
40.3
±0.33
39.3
±0.33
±S.E
15.3
±0.33
44.6
±0.33
43
±0.57
1
2
14.6±0.33
±
3
±
3
0.33 ±0.33 ±0.33 ±0.00
7.6 39.3 41 35.6
0.33 ±0.33 ±0.57 ±0.33
2.3 37.6 42 33.6
0.33 ±0.33 ±0.57 ±0.33
±0.33 ±0.33
45.6 30
±0.33 ±0.57
40 33.6
±0.57 ±0.33
38.6±0.33
35.3±0.33
CF
±
3
ILW- Topoisomerase II 2- Triazole substituted ciprofloxacin.

Design, Synthesis and Docking Studies of a Novel Ciprofloxacin 985
Table 2. Antibacterial activity of synthesized compounds.
Affinity
kcal/mol)
Dist from bestmode
Docking
Mode
(
Rmsd l.b
Rmsd u.b
1
2
3
4
5
6
7
8
9
1
2
3
4
5
6
7
8
9
-6.7
-6.7
-6.4
-6.1
-6.0
-5.8
-5.6
-5.6
-5.6
-6.2
-6.0
-5.8
-5.7
-5.6
-5.5
-5.4
-5.4
-5.4
0.000
2.867
2.236
3.942
3.537
0.000
5.004
3.799
4.862
6.614
3
ILW Vs Cipro
18.477
4.493
58.110
15.824
0.000
46.149
18.770
15.789
53.120
16.983
67.274
40.663
68.010
20.581
7.177
60.053
17.496
0.000
49.365
20.961
17.451
56.581
18.814
71.018
43.135
71.680
3
ILW Vs 2
Results and Discussion
The starting material thiocarbohydrazide was prepared according to the literature method by
reacting hydrazine hydrate and carbon disulphide. This was directly fused with the
carboxylic group of ciprofloxacin to get the triazole.The synthesized final compound was first
purified by successive recrystallization using appropriate solvents. The purity of the
synthesized compound was checked by performing thin layer chromatography and determining
1
melting points. IR and HNMR spectra were consistent with the assigned structure.
Molecular docking studies was employed for the analysis with training set composed of
our synthesized compound whose inhibitory activity is unknown, in order to find out the
molecular facilities responsible for biological activities. Ciprofloxacin and derivative of
ciprofloxacin (2) were taken for docking study. The compound used for docking showed
best fit (Root Mean Square Difference (rmsd) value of 0.000) with topoisomerase (3ILW)
protein. In order to determine the ability of autodock 4.0, to reproduce the orientation and
position of compounds in 3ILW protein, the standard and the derivative of ciprofloxacin
were extracted and docked back into the corresponding binding pocket. The compound
tested for docking study showed high affinity with low energy of -6.2 kcal/mol with
employed protein. Binding between 3ILW & compound indicates very good inhibition with
rmsd value of 0.000.Ciprofloxacin showed high affinity with low energy of -6.7 kcal/mol
with employed protein. Since our titled compounds are known to possess antimicrobial
activity, the compounds were screened for their antibacterial and antifungal activity by cup-
plate method. They were screened for antimicrobial activity against three bacterial strains (two
gram negative Klebsiella pneumoniae and Pseudomonas aeruginosa and one gram positive
Bacillus subtilis) and three fungal strains (Aspergillus niger, Rhizopus nigricans, Candida
albicans) by cup plate method (Table 3). Ethanol was used as control, showed no zone of
inhibition. The compounds were active against all the bacterial strains included in testing.

9
86 S. JUBIE et al.
Table 3. Antifungal activity of the synthesized compounds.
Com
Con
1
2
CF
C.albicans Conc, µg/mL
25, µg/mL ±S.E 50, µg/mL ±S.E 100, µg/mL ±S.E
-
-
-
-
-
-
48±0.00
70±0.00
36.6±0.
The triazole substituted ciprofloxacin showed good antibacterial activity against gram
negative bacteria but it was found to have moderate activity against gram positive bacteria
when compared to the standard.The titled compounds were tested for their antifungal
activity with three fungal strains (Aspergillus niger, Rhizopus nigricans, Candida albicans)
by using cup plate method (Table 6). All the fungal strains tested for assay showed
susceptible to all tested compounds, except Candida albicans. The triazole substituted
ciprofloxacin showed good antifungal activity against Candida albicans with zone of
inhibition of 70±0.00 mm.
1) 2-Chloroethanol
CS2
2NH -NH2
NH -NH-CS-NH-NH2
2
2
2
) NaOH, MWI,400 W
Carbon
disulphide
Thiocarbohydrazide
1
Hydrazine
hydrate
O
F
COOH
NH NHCSNHNH2
2
Thiocarbohydrazide
N
N
HN
reflux,3 hrs
Ciprofloxacin
N
O
N
N
SH
F
N
NH2
N
2
HN
Triazole substituted ciprofloxacin
Scheme
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