Design, Synthesis, Characterization, and Computational Studies on Benzamide Substituted Mannich Bases as Novel, Potential Antibacterial Agents

A series of benzamide substituted Mannich bases (1–7) were synthesized. The synthesized derivatives were authenticated by TLC, UV-Visible, FTIR, NMR, and mass spectroscopic techniques and further screened for in vitro antibacterial activity by test tube dilution method using amoxicillin and cefixime as standard drugs. The compounds 5, 6, and 7 were found to be the most active antibacterial agents among all the synthesized compounds. The physicochemical similarity of the compounds with standard drugs was assessed by calculating various physicochemical properties using software programs. The percent similarity of synthesized compounds was found to be good and compound 1 was found to have higher percentage of similarity. The compounds were subjected to QSAR by multilinear regression using Analyze it version 3.0 software, and four statistically sound models were developed with R 2 (0.963–0.997), R adj 2 (0.529–0.982), and Q 2 (0.998–0.999) with good F (2.35–65.56) values.


Introduction
The infectious diseases are widely managed by the antimicrobial agents but increase in resistance of microorganisms towards antimicrobial agents in the past few years has become a serious health care problem, and this has led to the necessity of designing of some novel, potent, and safe antimicrobial agents against resistant microbial strains. The compounds having Mannich bases are found to have broad spectrum activity against all strains resistant to other drugs. Mannich bases have gained importance due to their application in antibacterial activity [1][2][3].
Mannich bases are the end products of Mannich reaction with basic moiety of beta amino ketone [4,5]. Mannich bases are formed by the condensation of a compound with active hydrogen(s) with an amine (primary or secondary) and formaldehyde (any aldehyde) [6].
Computational studies are the starting point which can be used to predict the experimental data of entirely known molecule or to explore reaction mechanisms and so forth. There are several areas of computational studies and one of them is identification of correlations between chemical structures and properties and is known as QSAR. Quantitative structural activity relationship uses molecular parameters to quantify a pharmacological or chemical property for a set of molecules [7,8].
In the present work, novel series of seven benzamide substituted Mannich bases are prepared and further screened for the antibacterial activity against Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and Gram positive bacteria (Enterococcus faecalis, Staphylococcus aureus). Computational studies including QSAR and molecular structural similarity studies were also performed to establish a relationship between biological activities in terms of minimum inhibitory concentration with various physicochemical parameters using multilinear regression.

Material and Method
2.1. Chemistry. TLC plates of 3 × 15 cm coated with silica gel were used for reaction monitoring and for determination of retardation factor. Spots of TLC were located 2 The Scientific World Journal For mass spectra, solutions were made in HPLC grade methanol and spectra were obtained with Vg-11-250 J70S spectrophotometer at 70 eV using electron ionization (EI source). Chem3D Ultra (version 10) was employed for structural similarity studies [9]. QSAR studies were performed by multilinear regression using Analyze it version 3.0 software.

Synthesis.
A series of benzamide substituted Mannich bases were synthesized as per Figure 1.
General Procedure for the Synthesis of Benzamide. To 1 mL of benzoyl chloride, 2 mL of ammonium hydroxide (30%) was added. This mixture was allowed to stand for 2 minutes and then 4 mL water was added to it. The crude product was recrystallized with ethanol [10].

Biological Evaluation
3.1. Antibacterial Activity. The inhibition of the microbial growth may be utilized for demonstrating the therapeutic efficacy of the synthesized compounds. The Gram negative bacteria (Escherichia coli, Pseudomonas aeruginosa) and Gram positive bacteria (Enterococcus faecalis, Staphylococcus aureus) were used for the activity.
The antibacterial activity was evaluated by tube dilution method which depends upon the inhibition of growth of a microbial culture in a uniform solution of antibiotic in a fluid medium that is favorable to its rapid growth in the absence of the antibiotic [14]. In this method minimum inhibitory concentration (MIC) of the test compounds was determined. The MIC is the lowest concentration of an antimicrobial agent that inhibits the growth of the test organism [15]. Test compounds and standard compounds (amoxicillin and cefixime) were dissolved in dimethyl sulfoxide to give a concentration of 100 g/mL. Double strength nutrient broth I.P. was used. Suspension of each microorganism was made by transferring the organism from culture to 10 mL of sterile normal saline solution.

Determination of Minimum Inhibitory Concentration (MIC).
One mL of sterilized media was poured into sterile test tubes. One mL of 100 g/mL test solution was transferred in one tube and serially diluted to give concentrations of 50, 25, 12.5, 6.25, 3.12, 1.56, and 0.78 g/mL. To all the tubes 0.1 mL of suspension of bacteria in saline was added and the tubes were incubated at 37 ∘ C for 24 h. The growth in the tube was observed visually for turbidity and inhibition was determined by the absence of growth. MIC was determined by the lowest concentration of sample that retarded the development of turbidity. The activity of the compounds compared with the standard drugs (amoxicillin and cefixime) is given in Table 2. Graphical representation of minimum inhibitory concentration (MIC) of benzamide substituted Mannich bases against Gram positive and negative bacterial strains is given in Figures 2 and 3, respectively.

Molecular Structural Similarity Studies
Assessments of molecular structural similarity of synthesized benzamide substituted Mannich bases were compared to those of standard compounds by means of physicochemical The Scientific World Journal 5  similarity between the standard drugs and new analogues designed. The information was used for prediction of biological activity of important target compounds for novel drug discovery. The physicochemical parameters were calculated for the synthesized compounds using Chem3D Ultra (version 10) and compared with the values obtained for standard compounds (cefixime and tosufloxacin tosylate). Various set of parameters were used for calculations as given in Table 3. The distance of a particular target compound can be presented as where , is the value of molecular parameters for compound .
,standard is the value of the same molecular parameter for standard drug.
is the total number of the considered molecular parameters.
The similarity of the compounds can be calculated as  where is quadratic mean also known as the root mean square and can be calculated as Assessment of structural similarities of synthesized compounds with standard drugs showed that all the derivatives have good % age similarity (Table 4).

Quantitative Structure Activity Relationship (QSAR)
The synthesized substituted Mannich bases were subjected to QSAR analysis by using multilinear regression. For this analysis, various physicochemical parameters were calculated and correlated with biological activity, that is, antibacterial activity to obtain QSAR models. The physicochemical parameters were computed using Chem3D (Version 10) Ultra after energy minimization to minimum root mean square (RMS) gradient of 0.100 kcal/moleÅ by MOPAC software package. Out of all the physicochemical parameters (Table 4), 6 The Scientific World Journal the following five parameters were selected for QSAR studies: log , Connolly solvent accessible surface area (SAS), molar refractivity (MR), ovality, and molecular weight (MW). Biological activity data was converted to the logarithmic values. For antibacterial activity, biological activity was taken as −log(MIC/molecular weight of compound).

Statistical Analysis.
The statistical significance of the models was assessed on the basis of various parameters such as 2 (coefficient of correlation), 2 adj (coefficient of determination), 2 (cross validates 2 ), and F (Fischer statistics), considering all the parameters in the model significant only at 95% confidence level ( < 0.05).
Basic structure of synthesized benzamide substituted Mannich bases is shown in Scheme 1.
Here and hereafter, 2 : is coefficient of correlation, 2 adj : is coefficient of determination, : is Fischer statistics, : is number of test compounds, Press: is predictive error sum of squares, 2 : is cross validated 2 , BA: is biological activity, and : is standard error of estimation. The observed and predicted antibacterial activity of synthesized benzamide substituted Mannich bases are summarized in Table 5 and plot of calculated and observed antibacterial activity is given in Figure 4.
The observed and predicted antibacterial activity of synthesized benzamide substituted Mannich bases are summarized in Table 6 and plot of calculated and observed antibacterial activity is given in Figure 4.  in Table 7 and plot of calculated and observed antibacterial activity is given in Figure 5.
The observed and predicted antibacterial activity of synthesized benzamide substituted Mannich bases are summarized in Table 8 and plot of calculated and observed antibacterial activity is given in Figure 5.    to be comparatively similar to cefixime. While studying MIC against bacterial strains 7 was found to be most active among all the synthesized benzamide substituted Mannich bases. From the above results it was concluded that compounds bearing substitutions such as sulphonamido 5, p-nitro 6, and dinitro group 7 have emerged out as potent antibacterial agents.

Result and Discussion
The compounds bearing nitro groups were found to have significant potential against all bacterial strains. This activity was attributed to lipophilic nature of these substituents which ultimately facilitate the transportation of target compounds across the biological membranes to produce desired effect. Both nitro and sulphonamide groups are an efficient -I, -R group, but the nature of the latter (low log value) is more hydrophilic than the former (high log value). Hence the contribution of sulphonamide group towards antibacterial The results indicated that the bulkier aromatic substituents like sulphonamido group may increase lipophilicity and give better antibacterial activity. The presence of electron withdrawing group like -NO 2 in the compound resulted in enhanced biological activity by improving lipophilicity. This lipophilicity could facilitate penetration or passage of these compounds across the biological membrane easily for beneficial therapeutic effect.

Conclusion
In antibacterial activity, the compounds 5, 6, and 7 were observed as good antibacterial agents and compound 7 was found to be the most active against all the selected bacterial strains. The compound 1 was found to have excellent percentage of similarity (>90%) with all standard drugs. The results suggested that antibacterial activity is highly dependent on physicochemical parameters such as log , Connolly solvent accessible surface area (SAS), molar refractivity (MR), ovality, and molecular weight (MW). The derived model could be used in the future for designing of more potent inhibitors of bacterial infection.