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Abstract
The title compound TZ1 was
synthesized by N-alkylation reaction, and its structure was confirmed by 1H
NMR, 13C NMR and IR, it was screened for their in vitro antibacterial activity
by the agar well diffusion method against four bacteria, Gram-positive
(Bacillus cereus, Staphylococcus aureus) and Gram-negative (Escherichia coli,
Pseudomonas aeruginosa). The molecule was studied with the density functional
theory (DFT) at B3LYP/6–31G (d,p) level in order to determine the relationship
between the molecular structure and the antibacterial inhibition behavior. The
molecular geometry, frontier molecular orbitals and Mulliken atomic charge of
the compound are investigated to get a better insight of the molecular
properties. The molecular electrostatic potential (MEP) for a compound was
determined to check their electrophilic or nucleophilic reactivity. The
theoretical parameters offer significant assistance to understand the
antibacterial inhibition mechanism indicated by the molecule and are in full
agreement with the experimental results.
Keywords:5-Chlorosatin derivatives; N-alkylation reaction;
antibacterial activity; DFT; Molecular electrostatic potential
Introduction
The 5-Chloroisatin is well
documented as an important heterocyclic compound in the field of medicinal
chemistry. My recently published book and review [1,2] contain a special
chapter, dedicated to the chemistry of 5-Chloroisatin and their derivatives.
The 5-chloro-1H-indole-2 3-dione
structure is a heterocyclic compound which easily participates in chemical
reactions. Its bonding sites are analogous to pyrrole. As shown in Scheme 1,
5-Chloroisatin is reactive at four different positions including the carbon
atom 3, nitrogen atom 1, the C2–C3 p-bond and the C2–N sigma bond.
This moiety of 5-Chloroisatin and
their derivatives possess pharmacological and chemotherapeutic properties such
as anti- cancer [3], anti-diabetic [4], anti-inflammatory [5], anti-malarial
[6], anti-bacterial [7], anti-fungal [8], anti-viral [9] and others drugs for
treatment of several diseases [10].
Density functional theory (DFT) has
become a convenient method to decipher experimental results, in antibacterial
activity; this technique makes it possible to accurately predict the inhibition
efficiency of organic compounds on the basis of electronic and molecular
properties as well as reactivity indexes [11]. The objective of current study
is to explore relationship amongst structure and electronic properties of the
synthesized 5-chloro-1- (2- (dimethylamino) ethyl) indoline-2,3-dione (TZ1)
using DFT. Then, the evaluation of its antibacterial activity. (Scheme1)
Experimental Details
Chemistry
Melting points were determined using
the kofler bench apparatus and uncorrected. The spectra of 1H NMR spectra were
recorded in CDCl3 on the Brucker Avance-300 spectrometer, operating at 300MHz
and at 75MHz for 13C-NMR using TMS as an internal standard and Spin resonances
are reported as chemical shifts (d) in parts per million (ppm). Infrared
Spectra were run on AVATAR 320 AEK0200713 spectrometer and frequencies are
reported in cm-1. The purity of the synthesized compound was confirmed by thin
layer chromatography (TLC), performed on Silica gel 60 coated plates. UV light
was used for the visualization of TLC spots [12].
General
procedure
The 5-Chloroisatin derivatives TZ1
was prepared by mixing 0.2g (1.1 mmol) of 5-chloro-1H-indole-2,3-dione, (0.23g,
1.16mmol) of potassium carbonate in 15mL of N-N dimethylformamide (DMF) and
(0.035g, 0.10mmol) of BTBA, then, the reagent is slowly added, the mixture is
left at room temperature for 48 hours. The reaction mixture was concentrated by
using rota vapor. The solid was separated out by filtration. It was carefully
checked by thin layer chromatography. The compound was isolated by column
chromatography by using different fractions of n-hexane and ethyl acetate
[13-16].
Yield=89%; mp: 114 °C ; 1H NMR
(CDCl3) δppm 7.53-7.54 (m, H, HAr); 7.51 (d, H, HAr, J=9Hz); 6.90 (d, H, HAr,
J=9Hz); 3.85 (t, 2H, CH2, J=9Hz); 3.75 (t, 2H, CH2, J=9Hz); 2.15 (m, 6H, CH3).
13C NMR (CDCl3) δppm: 184.59 (C=O); 164.45 (N-C=O); 146.22, 141.13, 110.39
(Cq); 138.59, 126.08, 113.36 (CHAr); 55.90, 46.79 (CH2); 45.09 (CH3). Infra Red
(KBr) cm-1: 3565, 3174, 30815 (C-H), 2975, 1720 (C=O), 1607 (NC=O), 1445, 1472
(C=C) 1185,1123 (N-C), 654 (C-Cl).
Antibacterial
screening
Synthesized compound TZ1 was
screened for their antibacterial activity against two Gram positive (Bacillus
cereus and Staphylococcus aureus) and two Gram negative (Escherichia coli and
Pseudomonas aeruginosa) bacteria by the agar well diffusion method, using LB
medium (Luria Bertani medium: yeast extract 5.0g, peptone 10.0g, sodium
chloride 5.0g, distilled water 1000mL). This technique was recommended by CLSI
[17].
A sterile paper disk was placed on
the surface of each plate and impregnated with 5μL of the TZ1 solution at a
final concentration of 10mg/mL. Then, the plates were incubated at 4 °C for 2
hours to permit good diffusion before incubation at 37±2 °C for 24 hours. The
diameters of the inhibition zones were measured in mm with the caliper. A disc
impregnated with 2% dimethylsulfoxide as a negative control was made the
experiment was carried out in triplicate.
In order to determine the Minimum
inhibitory concentration (MIC) values, we started by the dilution of the TZ1
was prepared in a Mueller Hinton broth supplemented with bacteriological agar,
to reach a final concentration between 5mg/mL and 0.004mg/ mL, 50μL of
bacterial inoculum was added to each well at a final concentration of
106CFU/mL. DMSO (2%) was used as a negative control. The final concentration of
our product was between 5mg mL-1 (3rd well) and 0.019mg mL-1 (well 11). Plates
were incubated at 37 °C for 24 hours. After 2 hours of a subsequent incubation,
bacterial growth was revealed by reduction of blue dye resazurin to pink
resorufin [18].
Including, the minimum bactericidal
concentrations (MBC) which is the last step in the protocol, a bactericidal
control is carried out 24 hours earlier by streaking on a platelet agar, after
microdilution to the broth by spreading 5μL of the negative wells on Luria
Bertani agar plates.
Theoretical
calculations
The computational studies of
compound TZ1 were performed using the GAUSSIAN 09W [19] program package and
visualized with the Gauss View on a personal computer using density functional
theory (DFT) method with 6−31G (d,p) as the basis set [20]. The using HOMO and
LUMO orbital energies, the ionization energy and electron affinity can be
expressed as: IP = -EHOMO, EA = -ELUMO, respectively. The total hardness, η and
electronegativity χ were given by the following relations: 
[21].
Result and Discussion
Synthesis of
5-chloro-1-(2-(dimethylamino) ethyl) indoline- 2,3-dione (TZ1) is outlined in
Scheme 2, it was prepared according to a similar previous procedure [22].
5-chloroisatin was used as a starting material for the synthesis of various
substituted indole derivatives [23-27].
The (TZ1) was synthesized by the
N-alkylation reaction of 5-chloro-1H-indole-2,3-dione in DMF, a base K2CO3 and
a TBAB catalyst was added to a stirred solution at room temperature,
Chloro-N,N-dimethylethanamine was added dropwise to the mixture under
conditions of catalysis by phase transfer for 48 hours, the reaction was
monitored by thin layer chromatography. After this time, the mixture was
filtered and concentrated and dried under vacuum to afford the required
product. The complex was obtained in good yield, stable in air, and is colored
solid, soluble in methanol, chloroform, DMF, and DMSO.
The 1H-NMR, 13C-NMR and IR were used
to assign the structure of synthesized compound. (Scheme 2)
Antibacterial
activity
The In vitro antimicrobial screening
tests of synthesized compound TZ1 was carried out as an antibacterial activity.
the tested compound showed biological activity against different types of
Gram-positive (Bacillus cereus and Staphylococcus aureus) and Gram negative
bacteria (Pseudomonas aeruginosa, Escherichia coli), it showed zones of
inhibition of MIC/MBC values ranging from 0.156/0.156 to 0.313/0.313mm against
the Gram-positive bacteria and between 0.625/0.625 and 1.25/1.25mm against
Gram-negative bacteria.
Coordination enhances the
antibacterial activity and the TZ1 in the present study are more active against
Gram-positive than Gram-negative bacteria [28]. On the other hand, it should
also be noted that the presence of nitrogen and oxygen atoms which are the
highest values of the negative charge on the molecule TZ1 suggesting that these
centers have the maximum electron density and would preferentially interact
with the micro-organisms Gram positive then increases the antibacterial
potential.
Computational
details
Frontier orbital energy analysis and
other global reactivity descriptors: The all optimized structures along with
the numbering scheme of TZ1 at DFT/B3LYP level using the 6-31+G (d,p) basis are
shown in Figures 1-3.
The HOMO-LUMO orbitals help to characterize the chemical reactivity and
kinetic stability of the molecule.
The analysis of the HOMO highlights the areas of the molecule that can
donate electrons to electrophilic species while the analysis of the LUMO
predicts the regions of the molecule with high affinity to accept electrons
from nucleophilic species. The calculated HOMO–LUMO energy gap value is found
to be 3.1673 eV.
The dipole moment (μ(debye= 5.6982) tells about the polarity of the
molecule. The higher value of dipole moment in case of TZ1 molecule is mainly
attributed to an overall imbalance in charge from one side of a molecule to the
other side is also evident from the MESP plot. DFT calculation gives an idea
about the substance reactivity and site selectivity of the frameworks. By the
computed value of HOMO and LUMO energy values for the TZ1, the
electronegativity (χ), total hardness (η), Softness (σ), can be calculated. The
significance of (η) and (σ) is to evaluate both the reactivity and stability
[31].
Molecular electrostatic potential (esp) map: The molecular electrostatic
potential mapped surfaces show the charge distributions of molecules three
dimensionally which give clear and special signature of the interactions of the
compounds [31].
The molecular electrostatic potential is related to the electronic density
and a very useful descriptor for determining sites for electrophilic attack and
nucleophilic reactions as well as hydrogenbonding interactions [32].
The MEP mapped surface of the compound TZ1 was calculated by DFT/B3LYP at 6
31G (d,p) basis set and MEP surface are plotted in Figure 6. Red, blue and
green colors represent regions of the most electro negative, most electro
positive electrostatic and zero potential, respectively [33].
Mulliken charges analysis
The Mulliken atomic charges have a significant role in the application of
quantum chemical calculations to molecular systems, by determining the electron
population of each atom as defined by the basis function [34]. Table 3 exhibits
the calculated mulliken atomic charges except for atoms H by DFT/B3LYP at 6 31G
(d,p) basis set. Also, the color range in the scale of positive and negative
charge and graphical representation for Mulliken atomic charges of TZ1 is shown
in Figure 7.
From the listed tabulated values (Table 3) of atomic charge, we can summary
that the charge on the carbon atom (C6) is greater than other carbon atoms in
the all compounds because it is connected to electronegative chloride atom
(Cl10). Then, all nitrogen and oxygen atoms (N13, N22, O14 and O15) are the
most negatively charged ones, suggesting that these centers have the maximum
electron density, which can interact with the positively charged part of the
receptor easily.
Conclusion
In summary, we report the synthesis and characterization of 5-chloro-1- (2-
(dimethylamino) ethyl) indoline-2,3-dione (TZ1) in excellent yield. The
antibacterial activity of TZ1 has been explored experimentally and by quantum
calculations. The frontier orbital energy analysis, mulliken atomic charges and
electrostatic potential were also studied by using the DFT method at
B3LYP/6–31G (d,p). The antibacterial bioassay showed that it possessed
excellent activity.
Acknowledgements
The author would like to thank all the people who helped to carry out this
work such as 1H NMR, 13C NMR, IR, and for antibacterial activity.
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