الرئيسية / الفعاليات العلمية / بحوث / Design, Synthesis, and Antibacterial Study of New Gatifloxacin-Antioxidants as Mutual Prodrugs Noor H. Nasser, Sahar A. Hussein, Ahmed K. Hussein, Ammar A. Alibeg, and Zainab M. Jasim

Design, Synthesis, and Antibacterial Study of New Gatifloxacin-Antioxidants as Mutual Prodrugs Noor H. Nasser, Sahar A. Hussein, Ahmed K. Hussein, Ammar A. Alibeg, and Zainab M. Jasim

Received: 04 October 2019 / Received in revised form: 12 January 2020, Accepted: 14 January 2020, Published online: 28 February 2020
© Biochemical Technology Society 2014-2020
© Sevas Educational Society 2008
Abstract
Objective: The objective of this search is to design, and
synthesize new mutual prodrugs in order to obtain synergistic
antibacterial activity. Methods: The hydroxyl group of three
antioxidants (menthol, thymol, and vanillin), was linked to
chloroacetyl chloride through nucleophilic substitution reaction,
to give intermediates (Ia-Ic), which were reacted with a carboxyl
group of Gatifloxacin to form three final compounds (I-III)
having ester linkage. Results: The Antibacterial activity effect of
the target compounds (I-III) has been tested for in vitro inhibitory
activity against Gram-positive bacteria: Staphylococcus aureus
and Gram-negative bacteria: Escherichia coli by using spots
diffusion method. All the tested compounds show a remarkable
antibacterial activity against tested bacteria. Conclusion: The
synthesized prodrugs were characterized and identified through
FT-IR spectroscopy, 1H-NMR spectrum, and various
physicochemical parameters. The antibacterial study of the
compounds showed various activities toward the two types of
pathogenic bacteria which are Staphylococcus aureus and
Escherichia coli. Compounds [I, II, and III] revealed that
Staphylococcus aureus was sensitive to synthesized compounds
but Escherichia coli showed a reverse activity with some
resistance for antibacterial drugs .
Key words: Gatifloxacin, Antioxidants, Mutual Prodrugs,
Antibacterial activity
Introduction
Prodrugs are biologically inactive compounds that are activated
either chemically or enzymatically to give the active parent
components (Ratnadeep V. Ghadage, 2013). They are classified
to different classes according to the type of moieties (Zawilska et
al., 2013). The carrier linked prodrugs in which the drug molecule
(active moiety) was linked with carrier molecule (inactive
moiety), while the mutual prodrugs in the two active drug
molecules were linked to each other, either directly or through
specific linker reduce the steric factors between the two moieties
(Abu-jaish et al., 2014). Different bonds were used in the
synthesis of prodrug molecules as ester, amide…etc.), which
hydrolyzed by esterase and amidase enzymes respectively (Naser,
2018).
There are numerous objectives for prodrug synthesis such as
increasing water solubility, increasing oral bioavailability,
increasing chemical stability, reducing pain at injection site,
reducing side effects, increasing organs selectivity (Datar and
Shendge, 2015), and synergistic effect which is one of the
important objectives of mutual prodrugs like Benorilate (I) in
which the paracetamol (II) linked through ester linkage to aspirin
(III) in order to give synergistic analgesic effect, and reduce
gastric ulceration (Zhi-Z and Jiang, 2012).
Gatifloxacin (IV) is a fourth-generation fluoroquinolone
antibacterial agent (Sharma et al., 2009), which is a synthetic
compound that acts through suppression of bacterial DNA
synthesis by inhibition of gyrase or topoisomerase II enzyme in
gram-negative bacteria, and topoisomerase IV enzyme in grampositive
bacteria (Dougherty et al., 2014; Hawkey, 2003).
Fluoroquinolone possesses a broad antibacterial spectrum due to
its effect against gram-positive bacteria like Staphylococci, gramnegative
bacteria like Escherichia coli, Klebsiella, Serratia, and
Pseudomonas aeruginosa, anaerobic Chlamydia, Mycoplasma,
Legionella, Brucella, and Mycobacterium (Xia et al. 2013; Raul
et al., 2015).
Noor H. Nasser, Sahar A. Hussein, Ahmed K. Hussein,
Ammar A. Alibeg, and Zainab M. Jasim
Department of Pharmaceutical Chemistry, Faculty of
Pharmacy
, Kufa University, Iraq.
33 J Biochem Tech (2020) 11(1): 32-36
In order to increase its antibacterial activity, it was linked with
different anti-oxidants as menthol (V) (Leyva-López et al., 2017),
thymol (VI) (Waliwitiya et al., 2010), and vanillin (VII) (Kumar
et al., 2012), as they possess strong antibacterial activity, in
addition to their anti-oxidant, anticancer, and anti-inflammatory
properties (Chahal et al., 2017).
Materials and Methods
Experimental
The reagents and anhydrous solvents were of analytical grade and
supplied from (Reidal Dehean Germany; Sigma-Aldrich
Germany; BDH England). Melting points (uncorrected) were
determined by the capillary tube method by Thomas hover
apparatus (England). Rf values were determined through using
ascending thin layer chromatography, on DC-Kartan SI Alumina
0.2 mm to ensure the purity and progress of the reaction, using
methanol: benzene (50:50) as a mobile phase (Ahmed et al.,
2016). Determination of FT-IR spectra was done by using FT-IR
spectrophotometer and KBr discs, at the faculty of pharmacy,
Kufa University. Proton nuclear magnetic resonance (1H-NMR)
spectra were recorded using NMR ultra shield spectrophotometer
500 MHz, Bruker Avance III (Switzerland), at the college of
Tehran, Iran. Steps of synthesis of all compounds are presented in
scheme 1. Antioxidants (menthol, thymol, and vanillin) were
coupled with chloroacetylchloride in the presence of TEA, to give
intermediates Ia, Ib, and Ic. Then the coupling reaction of
Gatifloxacin with intermediates Ia, Ib, and IIc result in the
synthesis of compounds I, II, and III respectively.
Scheme 1: Synthesis of the compounds I, II, and III.
J Biochem Tech (2020) 11(1): 32-36 34
Chemistry:
Coupling reaction of antioxidants with chloroacetylchrolide:
Antioxidant (19.1 mmol), was dissolved in DMF: CHCl3 (25:75)
mixture (40 ml), then TEA (2.66 ml, 19.1 mmol) was added. The
reaction mixture was stirred on ice bath; chloroacetylchloride (1.5
ml, 19.1 mmol in 10 ml CHCl3) was added in dropwise with
continuous stirring over a period of one hour, followed by
refluxing of the mixture for three hours. Then excess cold water
was added, and the precipitated compound was filtered and
crystallized from ethanol, to give intermediate Ia, Ib, and Ic (Noor
et al., 2018). The percent yield, physical appearance, and Rf
values were given in Table 1.
Spectral Analysis:
2-isopropyl-5-methylcyclohexyl 2-chloroacetate (Ia); FT-IR
(cm−1): 2,972 (C-H) of alkane, 1,747 (C=O) of ester, and 1,226
(C-O) of ester.
2-isopropyl-5-methylcyclohexyl 2-chloroacetate (Ib); FT-IR
(cm−1): 2,970 and 2,939 (C-H) of alkane, 1,741 (C=O) of ester,
and 1,581 and 1,479 (C=C) of aromatic.
4-formyl-2-methoxyphenyl 2-chloroacetate (Ic); FT-IR (cm−1):
2,978 and 2,738 (C-H) of alkane, 1,789 (C=O) of aldehyde, and
1,685 (C=O) of ester.
Coupling reaction of Gatifloxacin with intermediates Ia, Ib,
and Ic.
A mixture of intermediated Ia, Ib or Ic (17.5 mmol), and
Gatifloxacin (17.5 mmol), were dissolved in DMF (25 ml), then
TEA (2.5 ml, 17.5 mmol) was added. The reaction mixture was
stirred at room temperature overnight. The solvent was
evaporated; the residue was triturated with acetone and
crystallized from methanol (Noor et al., 2018). The percent yield,
physical appearance, and Rf values were given in Table 1.
Spectral Analysis:
2-((2-isopropyl-5-methylcyclohexyl)oxy)-2-oxoethyl-1-
cyclopropyl-6-fluoro-8- methoxy-7-(3-methylpiperazin-1-yl)-
4-oxo-1,4-dihydroquinoline-3-carboxylate (I); FT-IR (cm−1):
3,048 (C-H) of aromatic, 2,978 and 2,850 (C-H) of alkane, 1,782
(C=O) of ketone, and 1,743 (C=O) of ester. 1H-NMR (DMSO-d6)
δ(ppm): 8.7 (s,1H,CH of alkene), 7.7 (s,1H,CH-Ar), 5.2 (s,2H,-
OCH2), 4.5 (m,1H,C-H), 4.1 (m, 1H, C-H of cyclopropane),3.8
(s,3H,OCH3), 3.5-2.6 (3m,7H, CH and CH2 of piperazine ring),
1.8-1.3 (m,13H,CH of alkane), 1.11 (d,3H,CH3), 1.08
(m,1H,NH), 0.88 (high intensity doublet, 9H,3CH3).
2-(2-isopropyl-5-methylphenoxy)-2-oxoethyl 1-cyclopropyl-6-
fluoro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylate (II); FT-IR (cm−1): 3,038 (CH)
of aromatic, 2,974 and 2,939 (C-H) of alkane, 1,732 (C=O) of
ketone, and 1,620 (C=O) of ester. 1H-NMR (DMSO-d6) δ(ppm):
8.7 (s,1H,CH of alkene), 7.7 (s,1H,CH-Ar), 7.2-7 (m,3H,CH-Ar),
5.2 (s,2H,-OCH2), 4.1 (m, 1H, C-H of cyclopropane),3.8
(s,3H,OCH3), 3.5-2.6 (3m,7H, CH and CH2 of Piperazine ring), 3
(m,1H,CH), 2.35 (s,3H,CH3-Ar), 1.3 (m,4H,2CH2 of
cyclopropane), 1.2 (high intensity doublet,6H,2CH3), 1.11
(d,3H,CH3), 1.08 (m,1H,NH).
2-(4-formyl-2-methoxyphenoxy)-2-oxoethyl 1-cyclopropyl-6-
fluoro-8-methoxy-7-(3-methylpiperazin-1-yl)-4-oxo-1,4-
dihydroquinoline-3-carboxylate (III); FT-IR (cm−1): 3,012 (CH)
of aromatic, 2,931 and 2,904 (C-H) of alkane, 1,732 (C=O) of
aldehyde, and 1,643 (C=O) of ester. 1H-NMR (DMSO-d6)
δ(ppm): 9.6 (s,1H,CH of aldehyde), 8.7 (s,1H,CH of alkene), 7.7-
7.3 (4d,4H,CH-Ar), 5.2 (s,2H,-OCH2), 4.1 (m, 1H, C-H of
cyclopropane),3.8 (s,6H,2 -OCH3), 3.5-2.6 (3m,7H, CH and CH2
of Piperazine ring), 1.3 (m,4H,2CH2 of cyclopropane), 1.11
(d,3H,CH3), 1.08 (m,1H,NH).
Antibacterial Study
The in vitro antibacterial activity of the synthesized compounds
was investigated against several pathogenic representative Grampositive
bacteria like Staphylococcus aureus and Gram-negative
bacteria like Escherichia coli by agar well diffusion method,
using Muell-Hinton agar as a medium (Ullah and Ali, 2017). All
bacteria used were obtained from the microbiology laboratory in
Middle Euphrates Hospital. 1ml of the spore suspension of each
bacterium was spread all over the surface of the cold solid media
placed in the petri-dish. The tested compounds were dissolved in
DSMO. An amount (0.1ml) of the solutions was added accurately
in spots on the surface of the injected solid media.
The Petri-dishes were incubated at37ᵒ C for 24house. The
inhibition zone formed by the compounds against the two types
from tested bacterial determined the antibacterial activities of the
synthetic compounds. The mean value obtained for two
individual replicates was used to calculate the zone of growth
inhibition of each compound.
Results and Discussion
Chemistry:
Mutual ester prodrugs of Gatifloxacin with antioxidants (menthol,
thymol, and vanillin) were synthesized according to the scheme
that was shown above. Their physicochemical characters were
represented in Table 1, and their structures were confirmed by the
FT-IR spectroscopy and 1H-NMR spectra. Anti-oxidants
(menthol, thymol, and vanillin) underwent nucleophilic
substitution reaction (SN2) in presence of equimolar of
chloroacetyl chloride when the hydroxyl group in their structures
attacked the electrophilic carbonyl carbon in chloroacetyl
chloride leading to the displacement of the chlorine atom. The
reaction occurred in the presence of equimolar of triethylamine
which acted as a base to neutralize the hydrogen chloride formed.
This reaction led to the formation of compounds Ia, Ib, and Ic.
35 J Biochem Tech (2020) 11(1): 32-36
The rate of an SN2 reaction follows second order kinetics, just
like how the rate of limiting step depends on the nucleophile
concentration as well as the concentration of the substrate (Smith
and March, 2007). This mechanism depends on the solvent,
temperature, and concentration of the nucleophile and that of the
leaving group. It is generally favored in primary or secondary
alkyl halides with an aprotic solvent like (DMF) (Marye Anne
FOX and James K. Whitesell, 2004). The synthesized compounds
undergo another nucleophilic substitution reaction with
Gatifloxacin, in which the secondary amine in the later compound
act as a nucleophile to attack the electrophile and cause
displacement of the chlorine atom from compounds Ia, Ib, and Ic.
In aliphatic heterocyclic compounds, the nitrogen atom is a part
of a saturated heterocyclic ring and the lone pair of electrons is
available for reaction with protons (e.g. Piperazine). In the base
strength compounds of this type are similar to their open-chain
aliphatic counterparts with typical pKa values of 8-9 (Donald,
2008).
Table 1: Physicochemical Properties of the Synthesized
Compounds.
Compounds
Empirical
Formula
Molecular
weight
Description
% Yield
Melting point
o C
Rf values
Ia C12H21ClO2 232.75
Deep brown
crystals
60.5 200-201 0.63
Ib C12H15ClO2 226.7
Pale yellow
powder
80 251-252 0.79
Ic C10H9ClO4 228.63
Pale Brown
crystals
84 202 d 0.8
I C31H42FN3O6 571.69 Brown Powder 76 198-200 0.72
II C31H36FN3O6 565.64
Pale yellow
Powder
62 289 d 0.62
III C29H30FN3O8 567.57 White crystals 63 184-186 0.84
Antibacterial activity:
The Antibacterial activity effect of the compounds has been
tested for in vitro growth inhibitory activity against Grampositive
bacteria: Staphylococcus aureus and Gram-negative
bacteria: Escherichia coli by using spots diffusion method
(Sonmez et al., 2019; Sharma et al., 2019). All the tested
compounds show a remarkable antibacterial activity against
tested bacteria. The results were listed in Table 2, and their
statistical results were shown in Figure 1. Compounds [I, II, and
III] revealed that Staphylococcus aureus was sensitive to
compounds but Escherichia coli showed a reverse activity with
some resistance to antibacterial drugs and this can be discussed
under four points:- 1-inhibition of cell membrane function, 2-
inhibition of cell wall 3-inhibition of nucleic acid and 4-
inhibition of protein synthesis (Ullah and Ali, 2017).
Table 2: Antibacterial activity data (zone of inhibition in
nm) of the compounds I, II, and III.
Compound
Bacteria
G(+Ve) G(-Ve)
S.aureus E.coli
S 15 10
I 23 16
II 21 17
III 20 11
Figure 1: Antibacterial activity data (Zone of inhibition in nm) of
the compounds.
Conclusion:
The designed compounds have been synthesized successfully as
shown in scheme 1 and their structures were confirmed, using
Fourier Transform Infrared Spectroscopy (FT-IR spectra), Proton
Nuclear Magnetic Resonance Spectroscopy (1H-NMR), and their
purity was confirmed by their physical data (melting points and
Rf values).
All the tested compounds show a remarkable antibacterial activity
against tested bacteria. Compounds [I, II and III] revealed that
Staphylococcus aureus was sensitive to synthesized compounds
but Escherichia coli showed a reverse activity with some
resistance to antibacterial drugs.
Acknowledgments:
We are grateful to the pharmaceutical chemistry department staff
in the Faculty of PharmacyUniversity of Kufa for providing
facilities to complete the synthesis of the target compounds and
their intermediates.
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