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| ORIGINAL ARTICLE |
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| Year : 2020 | Volume
: 4
| Issue : 4 | Page : 318-322 |
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Synthesis, characterization, and In vitro antibacterial activity and molecular docking studies of N4, N4'-dibutyl-3,3'-dinitro-[1,1'-Biphenyl]-4,4'-diamine
K Muddukrishnaiah1, V Vijayakumar2, B Samuel Thavamani3, VP Shilpa3, N Radhakrishnan2, Heba S Abbas4
1 Department of Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappalli, India
2 Department of Chemistry, Anna University, CEG Campus, Chennai, Tamil Nadu, India
3 Department of Pharmacy, Sanjo College of Pharmaceutical Studies, Palakkad, Kerala, India
4 Department of Microbiology, National Organization for Drug Control and Research, Giza, Egypt
| Date of Submission | 04-Apr-2020 |
| Date of Acceptance | 02-May-2020 |
| Date of Web Publication | 30-Dec-2020 |
Correspondence Address:
Mr. K Muddukrishnaiah
Department of Pharmaceutical Technology, Anna University, BIT Campus, Tiruchirappallu - 620 024, Tamil Nadu
India
 Source of Support: None, Conflict of Interest: None
DOI: 10.4103/bbrj.bbrj_52_20
Background: Resistant growth is recognized as a significant public health hazard to human health worldwide among the most critical bacterial diseases. The evolving multidrug-resistant species are now commonly found in community settings, not just in the hospital area, which means that antibiotic bacteria reservoirs are beyond the hospital. Aim: In this study, we synthesized novel N4, N4'-dibutyl-3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine from 3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine and evaluation of its antimicrobial activity against clinical bacteria. Methods: Single-step synthesis of novel N4, N4'-dibutyl-3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine from 3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine and well characterized using spectroscopic methods, namely FT-IR, NMR, mass spectrometry, and CHNS. Besides, prepared compound molecular docking investigations, molecular physicochemical, absorption, distribution, metabolism, and excretion (ADME) analysis were also carried out. Results and Discussion: Novel Synthesized N4, N4'-dibutyl-3,3'-diaminobenzidine (DAB) was conducted for antibacterial activity against clinical Klebsiella spp. and Staphylococcus aureus and Pseudomonas spp. by the disc-diffusion method and followed by serial dilution method. N4, N4'-dibutyl-3,3'-DAB showed bacteriostatic action of 500 μg/ml, 1000 μg/ml for Klebsiella spp. and Staphylococcus aureus. The molecular physicochemical investigation exhibited that 1 violation and ADME analysis presented a low gastro intestinal effect. Docking investigations disclosed the capability of synthesized molecule potential to dock with beta-lactamase protein through patch dock methodology. Conclusion: N4, N4'-dibutyl-3,3'-DAB is the novel compound that was found to be attractive for the “drug hunters” as a potential agent for the management of infectious diseases against the human pathogens Klebsiella spp. and Staphylococcus aureus.
Keywords: 1'-biphenyl]-4, 3'-dinitro-[1, 4'-diamine, beta-lactamase, in vitro studies, molecular docking, N4, N4'-dibutyl-3
How to cite this article:
Muddukrishnaiah K, Vijayakumar V, Thavamani B S, Shilpa V P, Radhakrishnan N, Abbas HS. Synthesis, characterization, and In vitro antibacterial activity and molecular docking studies of N4, N4'-dibutyl-3,3'-dinitro-[1,1'-Biphenyl]-4,4'-diamine. Biomed Biotechnol Res J 2020;4:318-22 |
How to cite this URL:
Muddukrishnaiah K, Vijayakumar V, Thavamani B S, Shilpa V P, Radhakrishnan N, Abbas HS. Synthesis, characterization, and In vitro antibacterial activity and molecular docking studies of N4, N4'-dibutyl-3,3'-dinitro-[1,1'-Biphenyl]-4,4'-diamine. Biomed Biotechnol Res J [serial online] 2020 [cited 2023 Mar 28];4:318-22. Available from: https://www.bmbtrj.org/text.asp?2020/4/4/318/305643 |
| Introduction | |  |
3,3'-diaminobenzidine (DAB) is monomer for the synthesis of polybenzimidazole-based polymeric resins, fiber, and proton-exchange membrane. DAB is derivatives of benzidine, which is utilized for the staining of nucleic acids and cells. The benzidine precursor molecule can intermingle with DNA by groove the binding and unfinished intercalation is well known.[1] 3, 3'-DAB and alkylated DAB have effectively utilized for biological staining of PCR-based DNA analysis.[2],[3] The target molecule N4, N4'-dinitro-3,3'-dibutylaminobenzidine is an intermediate for N4, N4'-dibutyl-3,3'-DAB monomer preparation.[4] Bioinformatics methods are nowadays replacing and are complementary to wet laboratory experiment in studying the structure and function of biological molecules. Molecular docking is one of the bioinformatics tools to study the interaction between the small molecules (ligands) and protein/enzyme (receptor), predict the binding site of the ligand.[5],[6] Fischer proposed the method for elucidating the interaction between the ligand and receptor, which was popularly called as Lock and Key theory, where both are treated as rigid bodies. Later, Koshland developed a theory called induced fit, where both ligand and receptors are treated as flexible bodies, in which the active site of the protein is continuously reshaped while interacting with the ligand.[7]
β-lactamases are the most important enzyme that confer drug resistance among Gram-negative bacteria. β-lactamases are enzymes that cleavage the β-lactam ring, and they can be encoded on extrachromosomal elements.[8] Incessant mutations in β-lactamases make them extremely diverse. β-lactamases, the key resistance determinant for β-lactam antibiotics in Gram-negative bacteria, are ancient enzymes whose origins can be traced back millions of years ago.[9] The finest of authors familiarity and literature review there was no literature for synthesis, characterization and in vitro-in vivo investigation of N4, N4'-dinitro-3,3'-dibutylaminobenzidine. The present study, target molecule was synthesized and characterized using spectroscopic methods namely, FT-IR, NMR, mass spectrometry, and CHNS analysis. The prepared molecule physiochemical, drug likeness, absorption, distribution, metabolism, and excretion (ADME) analysis and molecular docking studies with beta-lactamase were done. Furthermore, the in vitro studies of target molecule with beta-lactamase were investigated. In this study, single-step synthesis of novel N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine from 3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine. The antibacterial activity of prepared novel compound conducted against to clinical pathogenic Klebsiella spp., Staphylococcus, and Pseudomonas.
| Materials and Methods | | |
Chemicals and instruments
The analytical reagent grade of chemicals was used in the present study without further purification. 3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine synthesized from our lab, 1-bromobutane (Aldrich), Potassium carbonate (Aldrich), N, N'-Dimethylformamide (Aldrich), Ethyl acetate, Petrolium ether (Aldrich).
1H NMR and13C NMR spectra of prepared molecule were recorded onto the Bruker AVANCE 300 FT-NMR in Deuterated chloroform as solvent. Electrospray ionization mass spectrophotometry (ESI-MS) was logged on the water Q-TOF Premier mass spectrophotometer. FT-IR spectrum was recorded onto a Bruker Vector 22 FT-IR spectrophotometer in the region of 4000–400 cm-1 utilizing the KBr pellet methodology.
Synthesis of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine
N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine was synthesized by the alkylation of 3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine (1) (Scheme 1). The synthetic procedure involves 3, 3'-dinitro- [1, 1'-biphenyl]-4, 4'-diamine (5.480 g, 20 mmol) was taken for dried 100 mL round bottom flask containing N, N'-dimethylformamide and K2CO3 (5.5 g, 40 mmol) was added at 0°C; the stirring was sustained for additional 15 min followed by 1-bromobutane (6.44 mL, 60 mmol) was adeed drop-wise. The reaction mixture was stirred for further 7 h at ambient temperature. Consequently, the reaction mixture was slaked with ice cold H2O and separated with dichloromethane. The organic phase was dried over anhydrous Na2SO4. The target molecule was cleansed by adopting column chromatography (1:9 ratio of ethyl acetate/Hexane) methods and getting pure N4, N4'-dibutyl-3, 3'-dinitro- [1, 1'-biphenyl]-4, 4'-diamine (2).
Characterization of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine
Red color powder, 87% yield; m. p. 143-145°C. FT-IT (KBr Pellets) (cm-1) 3375, 3099, 3057, 2961, 2933, 2863, 1627, 1268, 1558, 1461, 1406, 1517, 1351.1H NMR (300 MHz, CDCl3): δ (ppm) 0.93 (t, J = 6 Hz, 6H); 1.41-1.56 (m, 4H); 1.64-1.70 (m, 4H); 3.29 (t, J = 3 Hz, 4H); 6.87 (d, J = 6 Hz, 2H); 7.62 (d, J = 6 Hz, 2H); 8.04 (t, J = 3 Hz, 2H); 8.29 (s, 2H).13C NMR (75 MHz, CDCl3): δ (ppm) 13.8, 20.6, 31, 42.9, 114.6, 123.4, 126.1, 131.8, 134.1, 144.7. ESI-MS Calcd 386.20; Found [M + 1] is 387.24. CHNS: Formula (C20H26N4O4), C, 62.16%; H, 6.78%; N, 14.50%; O, 16.56%. Found: C, 62.10%; H, 6.82%; N, 14.53%; O, 16.60%.
Strain, Culture media and sterile discs
N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine and standard drug were conducted for bacterial activity against to clinical Staphylococcus aureus, Pseudomonas spp., and Klebsiella spp. Microbial cultures procured from the government medical college from Tiruchirappalli, Tamil Nadu. Media used for microbial test was Muller-Hinton agar media of Himedia Pvt. Bombay, India. Sterile discs used for antimicrobial activity procured from Himedia Pvt. Bombay, India.
Antibacterial activity
Antibacterial activity of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine was studies by using disc-diffusion method. Staphylococcus aureus, Pseudomonas spp., and Klebsiella spp. inoculums were prepared by using nutrient broth media.[10],[11] Double-strength sterile Mueller Hinton agar media were prepared by autoclaving 7.6 g in 100 ml. Inoculate the test microorganisms on the Mueller Hinton agar plates by using sterile cotton swabs and wells make on Mueller Hinton agar plates by using borer. Dimethyl sulfoxide dissolved N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine were placed on sterile discs. Discs were dried aseptically under laminar air flow to remove the solvents. Dried discs are placed on the surface of culture inoculated Mueller Hinton agar plates. Plates are incubated for 30 min at the refrigerator to diffuse the N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine into the agar plate and finally plates were incubated at 37°C for 24 h. Antibacterial activity was evaluated by using Himedia zone reader.
Minimum inhibitory concentration
The minimum inhibitory concentration of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine was determined by broth macrodilution using sterile glass test tubes containing Muller-Hinton agar broth. The N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine concentration ranges tested were: 100-2000 μg/ml and Cefixime concentration range were 2000–3.906 μg/ml [Table 2]. Antimicrobial dilution were prepared and freshly diluted on the day of testing. The test was performed in duplicate.[11] | Table 1: Antibacterial activity of N4, N4'-dibutyl-3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine by disc diffusion method
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 | Table 2: Broth micro dilution assay of N4,N4'-dibutyl-3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine against clinical isolates of Klebsiella spp, Staphylococcus and Pseudomonas
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Insilco studies
Docking studies was employed utilizing Patch Dock online server while the binding site analysis was carried out using PyMOL software. Molecular physicochemical and drug-likeness property analysis of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine was investigated using a Molinspiration tool. Furthermore, ADME analysis of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine utilizing Swiss ADME software.[12]
Target protein preparation
The three-dimensional structure of Beta-lactamase (PDB ID: 3S1Y) was originated from the Research Collaboratory for Structural Bioinformatics Protein Data Bank. The obtained protein was prepared using UCSF Chimera software for removing ligands, water particles and metal ions (water without hydrogen bonds) were removed, and the prepared protein was saved in PDB format.[13]
| Results and Discussions | | |
Characterization of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine
The peak at 3375 cm-1 as a results of NH stretching frequency of 2 and 3099, 3057, and 2961, 2933, 2863 cm-1 as a results of aromatic and aliphatic CH stretching frequencies of 2. The peaks at 1627 and 1268 cm-1 due to NH bending vibration and C-N stretching vibrations of 2. The peaks at 1558, 1461, and 1406 cm-1 as a results of aromatic C = C stretching frequency of the synthesized molecule 2. The1H NMR chemical shift value 8.29 corresponding to NH proton of synthesized molecule 2. The aliphatic butyl group protons are appeared in 0.92–3.30 ppm and aromatic protons are observed at 6.86-8.05 ppm. The calculated ESI-MS value of synthesized molecule is 386.20; Found is [M + 1] is 387.24. CHNS: Formula (C20H26N4O4), C, 62.16%; H, 6.78%; N, 14.50%; O, 16.56%. Found: C, 62.10%; H, 6.82%; N, 14.53%; O, 16.60%. In addition, the synthesized molecule melting point was found to be 143-145°C.
Antibacterial activity of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine
Antibacterial activity of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diami ne was studied by discs-diffusion method, the outcomes are shown in [Table 1] and [Figure 1]. | Figure 1: Synthesis of N4, N4'-dibutyl-3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine
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Minimum inhibitory concentration of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine by broth micro dilution method against to clinical isolates of Klebsiella spp., Staphylococcus, and Pseudomonas
In silco studies
Molecular docking studies
The choice of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine could be beneficial for receiving an information of the physiochemical and medication resemblance possessions of the aforementioned molecule beforehand finishing docking investigations. Lipinski's rule of five was linked for receiving a comprehension of the overhead said possessions and for further assistance in the assurance of whether a lead molecule containing a specific pharmacological and biological activity could be finished into a verbally active medicine for human. Violation of the Lipinski's rule of five occurred when log A >5, MW >500, number of N, O (hydrogen bond receptor) >10, number of OH and NH (hydrogen bond donor) >5 and number of the rotatable bond (rotb) >15. In the present investigation, N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine exhibited one violation with respect to Lipinski's rule of five [Table 3]. | Table 3: Molecular physicochemical descriptors analysis of N4, N4'-dibutyl-3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine using Molinspiration online software tool
Click here to view |
With respect to the drug-likeness score, at was active when the score was >0, moderately active when it was-5.0 to -0.0 and <-5.0 where inactive. N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamineexhibited moderately active bioactivity score toward two descriptions as show [Table 4]. ADME forecast was mandatory beforehand carry out docking investigations this is normally recognized in the initial stages of drug finding, drug screening, and drug design, owing to its individual characteristics. [Table 5] displays the ADME profile of N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine was foretold as low gastrointestinal absorption influence; furthermore, this molecule also forecast to all the cytochrome P450 inhibition (except * CYP2D6 and * CYP1A2). | Table 4: Drug-likeness property analysis of N4, N4'-dibutyl-3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine using Molinspiration online software tool
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 | Table 5: Absorption, distribution, metabolism, and excretion analysis of N4, N4'-dibutyl-3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine using Swiss absorption, distribution, metabolism, and excretion online tool
Click here to view |
In the present study, N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diaminewith beta lactamase interaction are presented in [Table 6] and. The synthesized molecule was effectually bind with selected β-lactamase protein and the atomic contact energy was found to be-181.12. Amongst the amino acids in β-lactamase protein Asn 373 amino acid residue was interacted with the synthesized molecule through amino acid oxygen (Nitro group), and the bond distance was found to be 2.9 A°. To the best of our literature survey, there was no articles were presented for enzyme inhibitory actions of prepared molecule N4, N4'-dibutyl-3,3'-dinitro- [1,1'-biphenyl]-4,4'-diamine till date. | Table 6: The molecular docking analysis of N4, N4'-dibutyl-3,3'-dinitro-[1,1'-biphenyl]-4,4'-diamine with beta lactamase using PatchDock online software
Click here to view |
| Conclusion | | |
The structure of prepared novel molecule was strong minded on the basis of the Fourier-transform infrared spectroscopy (FT/IR) and1H and13C NMR results. The FT-IR and1H NMR results confirm the structures of newly prepared molecule. The antibacterial activity of the prepared molecule was showed an excellent activity against Gram-negative clinical Klebsella spp. and moderate activity against to clinical Staphylococcus and Pseudomonas. From this study suggests that N4, N4'-dibutyl-3,3'-DAB molecule may be attractive for the “drug hunters” as a potential agent for the management of infectious diseases against the human pathogens Klebsiella spp.
Acknowledgments
The authors are thankful to the Center for Biotechnology and Phyto-Pharmacognosy Research, Coimbatore, for carrying out the antimicrobial studies.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]
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