Synthesis , X-Ray Crystal Structure Study , Hirshfeld Surface Analysis , and Biological Activity of N-( 2-amino-phenyl )-2-methyl-benzamide

The title compound crystallizes in monoclinic crystal system, with space group P21/c. The compound exhibits intermolecular interactions of the types N–H⋅ ⋅ ⋅N, C–H⋅ ⋅ ⋅O, and C–H⋅ ⋅ ⋅ π; intramolecular interactions of the type N–H⋅ ⋅ ⋅N. The intercontacts are also studied usingHirshfeld surface analysis.The compound showed no remarkable antibacterial activity when screened against two gram-negative and two gram-positive bacteria.


Introduction
The title compound is a benzamide derivative.The compound consists of an amide group bridged to a benzene ring to which a methyl is attached on one side and a phenyl ring to which an amino group is attached on the other side.Benzamides are derived from benzoic acid, which are slightly soluble in water and soluble in many organic compounds.

Experimental
2.1.Materials and Methods.Chemicals were purchased from Sigma Aldrich Chemical Corporation. 1H NMR spectra were recorded on a Bruker 400 MHz NMR spectrophotometer in DMSO-d 6 solvent and the chemical shifts were recorded in  (ppm) downfield from tetramethylsilane.Elemental analysis was done using Perkin Elmer 2400 elemental analyzer and results are within 0.4% of the calculated value.Infrared spectra were recorded on a Perkin Elmer spectrophotometer in the range of 400-4000 cm −1 .

X-Ray Diffraction.
A suitable white single crystal was selected to collect X-ray diffraction data.Data were collected on a Bruker Kappa Apex II single crystal X-ray diffractometer equipped with Cu   radiation and CCD detector [3].Crystal structure was solved by direct methods using SHELXS-97.
After locating all nonhydrogen atoms, the structure was refined by full-matrix least-squares method using SHELXL-97 [4].The obtained model was refined by isotropic thermal parameters later by anisotropic thermal parameter.Hydrogens were placed at chemically acceptable positions.156 parameters were refined with 1929 unique reflections which converged the residual () to 0.057.

In Vitro Antibacterial Activity.
As N-substituted benzamides and their derivatives have numerous biological activities, it is worthwhile to investigate the antibacterial activity of the newly synthesized compound.Antibacterial activity was examined against two grampositive bacteria, namely, Bacillus subtilis (MTCC number 121) and Staphylococcus aureus (MTCC number 7443), and two gram-negative bacteria, namely, Proteus vulgaris (MTCC number 742) and Escherichia coli (MTCC number 730).The bacterial strains were inoculated in nutrient broth and kept for overnight culture at 37 ∘ C. Minimum inhibitory concentration (mic) is the lowest concentration at which blue color of the dye (indicator) turns to pink color [5].MIC was determined by microbroth dilution method using resazurin (7-hydroxy-3H-phenoxazin-3-one 10-oxide) as an indicator.Resazurin is a blue nonfluorescent, nontoxic, oxidation-reduction indicator.This was performed on 96well microtiter plates [6].
For susceptibility testing the plates were prepared in duplicates.Nutrient broth of 50 L was distributed to all the wells.50 L compound was added to third and fourth wells.Serial dilution was performed from the fourth well till the concentration reaches 0.39 × 10 −2 mg/mL.Finally, 10 L of bacterial suspension was added to all the wells.The concentrations of the prepared solutions were as follows: 0.5 mg/mL, 0.25 mg/mL, 0.125 mg/mL, 0.625 × 10 −1 mg/mL, 0.3125 × 10 −1 mg/mL, 0.156 × 10 −1 mg/mL, 0.78 × 10 −2 mg/mL, and 0.39 × 10 −2 mg/mL.Blue color indicates that the compound inhibits the growth of the bacteria, whereas pink color indicates the bacterial growth.
Inoculated plates were incubated at 37 ∘ C for 24 hours.One hour before the end of incubation 10 L of resazurin was added to all the wells.The plates were incubated for another hour.The change in color was assessed visually.The MIC was recorded.

Elemental Analysis.
In order to confirm the chemical composition of the synthesized compound, carbon (C) and hydrogen (H) analysis was carried out.The experimental and calculated percentages of C and H are given in Table 1.The differences between experimental and calculated percentages of C and H were very small and are within the experimental errors.This confirms the formation of the product in the stoichiometric proportion.

FT-IR Spectral Analysis.
In FT-IR spectra the peaks observed at 1660 cm −1 are assigned to C=O of carbonyl of the amide group.The peak at 1715 cm −1 is for N-H stretching vibrations.2. The geometrical calculations were carried out using the program PLATON [7].The molecular and packing diagrams were generated using Mercury [8]. Figure 2 shows the ORTEP diagram of the molecule with thermal ellipsoids drawn at 50% probability.The bond   distances and angles are listed in Table 3. Torsion angles are listed in Table 4.
3.5.Hirshfeld Surface Analysis.CrystalExplorer 3.1 [10] program was used for understanding the interactions and the connectivity among the molecules efficiently.The crystallographic information file (.cif) was imported to the Crystal-Explorer to generate the Hirshfeld surfaces.The Hirshfeld surface is the region around the molecule in the crystal space which can be considered as the boundary separating two regions-the interior (the reference molecule) and the   The fingerprint plot shows the percentage contributions to the total Hirshfeld surface area.The fingerprint plot data is shown in Table 6.Figures 6(a 3.6.In Vitro Antimicrobial Activity.The results of biological activity of the title compound are given in Table 7.The resazurin assay showed that the compound has lower-toaverage activity against various tested bacterial strains.The results of the compound with tested bacterial strains were compared with streptomycin.Streptomycin was used as standard in the experiment.The compound showed better/average activity against gram-negative bacteria Proteus vulgaris than any other bacteria, though it never outperforms streptomycin.

Conclusion
In this research work we have discussed the synthesis of the compound N-(2-amino-phenyl)-2-methyl-benzamide.The preliminary characterizations like elemental analysis, FT-IR, 1 H NMR spectra of the compound are shown in Figure1.The NMR peak at  2.25 (s, 3H) clearly indicates that the three hydrogens of methyl group are attached to aromatic ring.The peak at  4.25 (bs, 2H) corresponds to the two hydrogens of the amino group.The peaks at  7.15-7.52refer to eight aromatic hydrogens of the compound.

Figure 1 :
Figure 1: 1 H NMR spectra of the title compound.

Figure 2 :
Figure 2: ORTEP diagram of the molecule with thermal ellipsoids drawn at 50% probability.

Figure 3 :
Figure 3: Packing of molecules when viewed along a-axis.

Figure 4 :
Figure 4: Packing of molecules when viewed along b-axis.

Figure 5 :
Figure 5:  norm mapped on Hirshfeld surface for the visualization of the intercontacts of the title compound.

Figure 6 :
Figure 6: Fingerprint plot of the title compound.(a) Highlighting full two-dimensional map.(b) Two-dimensional map resolved into H⋅ ⋅ ⋅ H contacts.(c) Two-dimensional map resolved into C-H/H⋅ ⋅ ⋅ C contacts.(d) All contacts.(e) Major H⋅ ⋅ ⋅ H contacts.(f) Major C-H contacts.

Table 1 :
Elemental analysis for the title compound.

Table 2 :
The crystal data and structure refinement details.

Table 6 :
Percentage of various intermolecular contacts contributing to Hirshfeld surface.

Table 7 :
MIC of the title compound against various bacterial strains.