Computational Analysis of a Series of Chlorinated Chalcone Derivatives

A systematic conceptual density functional theory (DFT) analysis was performed on a series of chlorinated chalcones to study the effect of electron distribution on antimicrobial activity. In our previous work, a series of 16 chlorinated chalcones were synthesized to determine the antimicrobial effects of varying the location of the halogen substituent on each aromatic ring of the chalcone. Herein is reported a DFT investigation of those 16 chalcones and a comparison of quantum chemical properties to their antimicrobial activity. DFT global chemical reactivity descriptors (chemical hardness/softness, chemical potential/electronegativity, and electrophilicity) and local reactivity descriptors (Fukui functions and dual descriptor) were calculated for all compounds using Spartan’18 software. All calculations were carried out at the B3LYP/6-31G* level of theory. Reactivity analysis of the Fukui dual descriptor calculations reveals sites of nucleophilic and electrophilic attack. These in-silico results provide a foundation for further synthetic optimization of the chalcone skeleton to serve as novel antimicrobial agents.


Introduction
Treatment of bacterial infection is a major health issue, with increasing emergence of drug-resistant microbes resulting in two million infections and over twenty thousand deaths each year in the United States alone [1]. Thus, it is crucial to develop new antibiotics to combat these pathogens. Chalcones, of the flavonoid family, are characterized by their dual aromatic rings and α,β-unsaturated ketone moiety which is responsible for their widely known antimicrobial activities [2]- [10]. In addition to antimicrobial activity, chalcones have been shown to exhibit a multitude of other pharmacological effects including, but not limited to, antioxidant [11] [12], anti-inflammatory [13] [14] [15], and antitumor [16] [17] [18] [19]. Synthesis and biological evaluation of chlorine positioning were previously studied in our laboratory [10]. To further the understanding of halogen placement on biological activity, an in-silico analysis can be employed.
Density Functional Theory (DFT) has been shown to elucidate valuable descriptions of chemical reactivity, molecular structure, vibrational frequencies, and the energetics of chemical reactions [20] [21] [22] [23] [24]. DFT has produced powerful tools for the study of organic reactivity via the use of theoretical global and local reactivity indices and is widely utilized by researchers in chemistry, physics and materials science due to the development of accurate density functionals. Novel DFT studies routinely provide insights into chemical reactivity [24] [25] but no such studies have been performed on chlorinated chalcones.
The goal of this computational analysis is to investigate the electronic properties of a series of 16 chalcones and compare them to their previously reported antimicrobial activity.

Computational Details
Quantum chemical calculations were performed using Spartan'18 computational software on a Macintosh computer. Geometry optimization and single point energy calculations were carried out at the B3LYP/6-31G* level of theory and verified by the absence of any imaginary frequencies.

Electronic Chemical Potential (μ) and Mulliken Electronegativity (χ)
The electronic chemical potential (μ) of a molecule is the change in energy with respect to the number of electrons (N) at a fixed external potential ( ) r υ [27] [28]. This represents the tendency of electrons to escape from an atom or molecule.
Application of the finite difference approximation yields the simplified expression: where IP represents the first ionization potential and EA represents the electron affinity of an atom or molecule. The Koopman theorem [29] and Kohn-Sham formalism [30] show that the ionization potential (IP) can be approximated by the energy of the highest occupied molecular orbital (−E HOMO ) and the electron affinity (EA) can be approximated by the energy of the lowest unoccupied mo- Given that electronegativity ( χ ) is characterized as the tendency of an atom to attract electrons, it can be written as the negative of the chemical potential ( µ ):

Chemical Hardness (η) and Softness (S)
The hard and soft acids and bases (HSAB) principle presented by Pearson [30] [31] [32] states that Lewis acids can be classified as either hard or soft [33] and Using the finite difference approximation and substituting the ionization potential (IP) for −E HOMO and electron affinity (EA) for −E LUMO , the chemical hardness can be expressed as: Chemical softness (S) is the reciprocal of chemical hardness (η):

Electrophilicity Index (ω)
Maynard [34] and Parr [35] defined the electrophilicity index (ω) as the tendency of an electrophile to acquire more electron density (chemical potential) divided by the resistance to exchange electron density with the environment (chemical hardness). It is a quantitative value intrinsic to a molecule and has become a powerful tool to predict reactivity.

Local Quantum Chemical Descriptors Fukui Functions and Dual Descriptor
The Fukui function ( ) Parr expanded the use of the Fukui function to show localized areas of nucleophilicity ( f + ) and electrophilicity ( f − ) called the condensed Fukui func- A condensed form is represented by:

Results and Discussion
Chalcones 1a-d, 2a-d, 3a-d, and 4a-d were previously synthesized and tested for antimicrobial activity in our laboratory [10] using agar well diffusion assay by measuring the zones of inhibition against pathogenic bacteria (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus). The results show that nearly all the chlorinated chalcones synthesized display antibacterial activity comparable to the known antibiotic sulfanilamide. The 16 chalcones, shown in Figure 1 below, vary the positioning of chlorine substituents on each ring of the chalcone skeleton. In order to probe the chemical rationale for the differences in biological activity, each chalcone was analyzed computationally.
Full unconstrained molecular geometry optimizations of 16 chalcones were carried out using Wavefunction Spartan'18 software [42] using the exchange correlation functional by Becke [43] with gradient-corrected correlation provided by Lee, Yang and Parr [44] with the 6-31G* basis set. This DFT method was chosen since it is fast, less computationally intensive, takes electron correlation into account, and provides accuracy in reproducing experimental data [45].    chalcones. It is also notable that Series 4 chalcones were the softest as well. The most electrophilic chalcone overall was 3c however.        (10) and (11) to determine the f + and f − Fukui indices respectively. The combined values according to equation 13 yields the dual descriptor, where f ∆ is a function that represents the reactivity of the atoms in each molecule. All 16 chalcones have negative values on C1 (carbonyl carbon) and C3 (terminal alkene carbon) which follows the logic predicted by modern resonance theory. C1 had a larger negative value than C3, representing a higher likelihood for nucleophilic attack to occur there. Chalcone 1d had the highest value on C1 while chalcone 1b has the highest value on C3. Chalcone 4d had the overall highest values for both atoms.
The four chalcones of Series 4 showed the highest antibacterial activity from our previous testing, with 4c showing the largest zone of inhibition of all 16 chalcones [10]. From our computational analysis, the chalcones of Series 4 were also the most electrophilic and softest. In addition, chalcone 4c had the highest averagezone of inhibition when taking into account all three bacterial strains evaluated and was the softest of all 16 chalcones. From these results, it can be determined that increasing electrophilicity and softness are vital for enhancing antimicrobial effectiveness.
Future studies will build on this discovery by synthesizing chalcones substituted in the 4-position and containing substituents that result in greater electrophilic nature. The high values of logP were also confirmed experimentally with less than ideal solubility in aqueous media. Choosing substituents that will reduce the lipophilicity should result in an enhanced bioavailability.

Conclusion
Using conceptual DFT, it was determined that chalcones with a chlorine in the 4-position (Series 4) generally contained the most electrophilic and softest chalcones. These were the most active chalcones in previous antimicrobial evaluation. Adding chlorine moieties increases lipophilicity regardless of the positioning, but none of the 16 chalcones exceeded a logP of 5. Having a chlorine atom in position 4 of the chalcone skeleton proves to be the most effective location. The results of this study enable further optimization of the chalcone skeleton by retaining the chlorine in position 4 while probing the effects of adding more electrophilic substituents.