3.1. The chemical synthesis outcome was produced through the manipulation of chemical reactions.
A groundbreaking report has detailed the synthesis of biaryl analogs through the utilization of Suzuki coupling reaction, which involves combining haloaryl with a range of phenylboronic acids. The study has led to significant advancements in the production of diverse biaryl derivatives through the use of Suzuki coupling reactions. Specifically, the research details the coupling reactions of various substituted bromobenzene with substituted phenylboronic acids (1a–b) to produce corresponding biaryls (3a–k), using the optimal reaction conditions of Pd(OH)2 as a catalyst, potassium phosphate as a base, and a temperature of 72°C. The use of equimolar substituted aryl-boronic acids 2a-e resulted in rational yields of biphenyl derivatives 3a–k, with greater yields achieved through the use of a solvent mixture of ethanol and water instead of toluene. The final yield of biphenyl derivatives was highly dependent on the choice of solvent used, with the best results achieved using a 2:5 (water/ethanol) solvent mixture. The reaction was shown to be both efficient and environmentally friendly, with no impact on yield observed at temperatures of 72°C and 55°C. The synthesized compounds were thoroughly characterized using spectroscopic evaluation techniques such as IR and 1H NMR, with the data obtained found to be in excellent agreement with the calculated values of the projected structures. Furthermore, preliminary testing revealed promising antimicrobial activity for the newly obtained compounds.
Scheme1. The coupling of aryl halide with arylboronic acid using the Suzuki-Miyaura reaction.
2-Fluoro-4'-nitrobiphenyl (3a)
1H NMR (400 MHz, DMSO-d6, ppm): δ 7.19 (t, J = 8.0 Hz, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.75 (d, J = 8.0 Hz, 2H), 8.33 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6, ppm): δ 116.0, 123.6, 124.1, 128.6, 131.2, 143.1, 147.8, 161.1.LC-MS m/z 218 (M + 1).
2,4'-Difluoro-3'-(trifluoromethyl)biphenyl (3b)
1H NMR (400 MHz, DMSO-d6, ppm): δ 7.23 (t, J = 8.0 Hz, 2H), 7.45 (t, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz, 1H), 7.84 (s, 1H). 13C NMR (100 MHz, DMSO-d6, ppm): δ 111.2, 116.8, 118.4, 124.1, 124.6, 130.1, 132.9, 157.3, 160.4. LC-MS m/z 259 (M + 1).
2,4'-Difluoro-3'-(trifluoromethoxy)biphenyl(3c)
1H NMR (400 MHz, DMSO-d6, ppm): δ 7.26 (t, J = 8.0 Hz, 2H), 7.45 (d, J = 8.0 Hz, 2H),7.52 (d, J = 8.0 Hz, 2H), 7.49 (s, 1H). 13C NMR (100 MHz, DMSO-d6, ppm): δ 114.6, 116.0, 117.0, 122.3, 124.6, 129.0, 133.2,146.6, 149.5, 161.0. LC-MS m/z 275 (M + 1).
2,2'-Difluoro-3-nitrobiphenyl(3d)
1H NMR (400 MHz, DMSO-d6, ppm): δ 7.21 (d, J = 8.0 Hz, 1H), 7.32 (t, J = 8.0 Hz, 2H), 7.48 (d, J = 8.0 Hz, 2H), 7.72 (d, J = 8.0 Hz, 1H), 8.15 (d, J = 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6, ppm): δ116.0, 124.1, 125.5, 128.3, 131.5, 135.3, 154.9, 163.4. LC-MS m/z 236 (M + 1).
2,2'-Difluoro-5-methoxybiphenyl (3e)
1H NMR (400 MHz, DMSO-d6, ppm): δ 3.74 (s, 3H), 6.71 (d, J = 8.0 Hz, 1H), 6.92 (d, J = 8.0 Hz, 2H), 7.20 (t, J = 8.0 Hz, 2H), 7.47 (d, J = 8.0 Hz, 1H), 13C NMR (100 MHz, DMSO-d6, ppm): δ54.6, 111.0, 121.5, 125.1, 131.8, 135.9, 155.2, 161.6. LC-MS m/z 221 (M + 1).
2-Methoxy-4'-nitrobiphenyl (3f)
1H NMR (400 MHz, DMSO-d6, ppm): δ 3.71 (s, 3H), 7.15 (t, J = 8.0 Hz, 1H), 7.28 (t, J = 8.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 1H). 7.67 (d, J = 8.0 Hz, 2H), 8.16 (d, J = 8.0 Hz, 2H). 13C NMR (100 MHz, DMSO-d6, ppm): δ55.4, 114.5, 121.5, 128.7, 129.8, 135.9, 147.5, 155.2, 159.7. LC-MS m/z 230 (M + 1).
4'-Fluoro-2-methoxy-3'-(trifluoromethyl)biphenyl (3g)
1H NMR (400 MHz, DMSO-d6, ppm): δ 3.81 (s, 3H), 7.13 (t, J = 8.0 Hz, 1H), 7.22 (t, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0 Hz, 1H), 7.48 (d, J = 8.0 Hz, 1H). 7.81 (d, J = 8.0 Hz, 2H), 7.95 (d, J = 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6, ppm): δ54.8, 108.4, 112.2, 114.5, 118.3, 121.5, 125.5, 131.2, 145.8, 161.0. LC-MS m/z 270 (M + 1).
3'-Fluoro-2-methoxy-5'-(trifluoromethyl)-1,1'-biphenyl (3h)
1H NMR (400 MHz, DMSO-d6, ppm): δ 3.76 (s, 3H), 7.23 (t, J = 8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 1H), 7.31 (s, 1H), 7.37 (d, J = 8.0 Hz, 2H), 7.52 (d, J = 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6, ppm): δ54.8, 113.4, 114.2, 121.6, 125.1, 128.3, 133.4, 142.5, 149.7. LC-MS m/z 287 (M + 1).
2-Fluoro-2'-methoxy-3-nitrobiphenyl(3i)
1H NMR (400 MHz, DMSO-d6, ppm): δ 3.65 (s, 3H), 6.89 (d, J = 8.0 Hz, 1H),7.35 (t, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.77 (d, J = 8.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6, ppm): δ54.9, 114.5, 121.4, 124.1, 128.6, 134.7, 155.3, 162.5. LC-MS m/z 248 (M + 1).
2-Fluoro-2',5-dimethoxybiphenyl (3j)
1H NMR (400 MHz, DMSO-d6, ppm): δ 3.69 (s, 6H), 6.78 (d, J = 8.0 Hz, 1H), 6.97 (s, 1H), 7.11 (t, J = 8.0 Hz, 2H), 7.23 (d, J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.61 (d, J = 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6, ppm): δ54.9, 113.6, 122.7, 126.0, 128.5, 153.6, 161.6. LC-MS m/z 233 (M + 1).
2'-chloro-2-fluoro-5-methoxy-1,1'-biphenyl (3k)
1H NMR (400 MHz, DMSO-d6, ppm): δ 3.84 (s, 3H), δ 7.21–7.39 (s, 5H), 7.69 (s, 1H), 7.21 (t, J = 8.0 Hz, 2H), 7.34 (d, J = 8.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6, ppm): δ56.19, 113.82, 114.17, 117.90, 128.5, 130.0, 133.06, 137.22, 151.11, 156.12. LC-MS m/z 238 (M + 1).
3.2. In silico toxicity studies
In silico toxicity assessments utilize computational methods to predict the potential toxicity of a chemical compound. They can be used in drug development and to evaluate the safety of substances that come into contact with humans or the environment. These assessments are a valuable tool for predicting and evaluating the potential risks of chemical substances.
3.2.1. Comprehensive Assessment of the ADMET Properties of Compound 3f
The results presented in Table 1 provide a comprehensive assessment of the ADMET properties of the compound 3f. This information is valuable in guiding further research and development towards identifying potential drug candidates with desirable properties. The table includes information on a range of molecular properties, including the compound's molecular formula, molecular weight, number of heavy atoms, aromatic heavy atoms, rotatable bonds, and hydrogen bond acceptors. The table also assesses the compound's potential as a GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor, and enzyme inhibitor. The results show that the compound 3f has zero Lipinski violations, indicating that it is likely to be able to cross cell membranes and enter cells effectively. The compound also has moderate potential as a GPCR ligand, and low potential as an ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor, and enzyme inhibitor. In terms of its molecular properties, the compound 3f has moderate lipophilicity and moderate to high polarity and potential for hydrogen bonding. The compound also has moderate potential for absorption and availability, and moderate solubility. Overall, these results provide a valuable overview of the ADMET properties of the compound 3f, which can be used to guide further research and development towards identifying potential drug candidates with desirable properties. The information presented in Table 1 is important in assessing the potential efficacy and safety of the compound 3f as a drug candidate, and highlights the need for careful consideration of molecular properties and ADMET characteristics in drug development. Moreover, Drug-likeness model score value 1.33 which suggests that the compound being evaluated has a high drug-likeness score. This score indicates that the compound 3f is likely to have drug-like properties in terms of its physicochemical and structural properties. Further studies may be warranted to determine the potential of this compound as a drug candidate. Olso, based on the information provided in Fig. 1, the compound 3f has a probability of being bioactive against the listed protein targets. These results contribute to a growing body of research aimed at identifying safe and effective drug candidates for a range of diseases and conditions.
Table 1
The key ADMET characteristics of the compound 3f
Properties
|
Molecule
|
Compound 3f
|
Formula
|
C13H11NO3
|
MW
|
229.24
|
#Heavy atoms
|
17
|
#Aromatic heavy atoms
|
12
|
#Rotatable bonds
|
3
|
#H-bond acceptors
|
3
|
#H-bond donors
|
-
|
MR
|
67.19
|
TPSA
|
55.05 Ų
|
Consensus Log P
|
3.53
|
Ali Class
|
Moderately soluble
|
Silicos-IT LogS
|
-4.47
|
Silicos-IT class
|
-4.43
|
log Kp (cm/s)
|
-5.12 cm/s
|
Lipinski #violations
|
Yes; 0 violation
|
Bioavailability Score
|
0.55
|
GPCR ligand
|
-0.45
|
Ion channel modulator
|
-0.13
|
Kinase inhibitor
|
-0.41
|
Nuclear receptor ligand
|
-0.33
|
Protease inhibitor
|
-0.67
|
Enzyme inhibitor
|
-0.23
|
Table 2
The compound 3f toxicity prediction using ADMET SAR
Model
|
compound 3f
|
Blood-Brain Barrier
|
Result
|
BBB+
|
Probability
|
4.52
|
CYP Inhibitory Promiscuity
|
Result
|
High CYP Inhibitory Promiscuity
|
Probability
|
0.3062
|
Human Intestinal Absorption
|
Result
|
HIA+
|
Probability
|
1.012
|
Caco-2 Permeability
|
Result
|
Caco2+
|
Probability
|
0.6708
|
Carcinogenicity (Three-class)
|
Result
|
Non-required
|
Probability
|
0.3245
|
Acute oral toxicity
|
Result
|
II
|
Probability
|
0.4839
|
Rat Toxicity LD50 mol/kg
|
Result
|
1.2618
|
3.3. Docking results
3.3.1/ Molecular Docking Analysis of Proposed Compounds with E. coli Fabh: NCBI's PDB Repository and MOE's Role in Structural Biology
The National Center for Biotechnology Information (NCBI) is an integral component of the United States National Library of Medicine (NLM), which operates as a subsidiary of the National Institutes of Health. The NCBI offers an extensive Molecular Modeling Database that serves as a critical isomer in the field of structural biology. It functions as a crucial repository for 3D structure data of large molecules, contributing immensely to the development of molecular research. The sequence with pdb id: 5bnr was extracted from the protein database at the NCBI, which can be accessed at https://www.rcsb.org/structure/5BNR. To perform the docking studies of the proposed compounds, the Molecular Operating Environment (MOE) was employed. The aim was to explore the molecular details of the mode of interaction with E. coli Fabh (Beta-ketoacyl-[acyl-carrier-protein] synthase III), and the proposed compounds were docked into the binding site with a cocrystallised ligand (the PDB codes for the crystallographic structure is (5bnr)). The potent compound exhibiting the lowest RMSD (root-mean-square deviation) values compared to the crystal structures was analyzed. In each case, the scale of the score served as a guide to determine the effectiveness of the docking pose, with a higher score indicating a better docking result. By using smart minimizer algorithm with a maximum number of steps 1000 and RMS gradient 0.1, energy minimization is performed that carry forward using steepest descent and conjugate gradient algorithm till the protein complex makes a grade of convergence gradient 0.0011 kcal/mol. This analysis enables a better understanding of the molecular interactions and assists in the design of more effective compounds with better docking scores for E. coli Fabh as in Fig. 2.
3.3.2. Refinement of E. coli Fabh Protein Crystal Structure using Restrained Minimization and Force Field Analysis
In this study, the crystal structure of E. coli Fabh protein (pdb id: 5bnr) was downloaded from the Protein Data Bank and subjected to a refinement process. The refinement involved correcting bond orders, formal charges, and missing hydrogen atoms, as well as topologies and incomplete and terminal amide groups. Water molecules were removed, and the ionization states of heteroatoms were generated and chosen. Hydrogen bonds were assigned, and the orientations of retained water molecules were corrected. Finally, a restrained minimization of the protein structure was carried out using a force field to reorient side-chain hydroxyl groups. The minimization was restricted to the input protein coordinates by a predefined Root Mean Square Deviation (RMSD) tolerance of 0.3 Å. Figures 3 and 4 represent the crystal structure of E. coli Fabh (pdb id: 5bnr). Moreover, the physicochemical attributes of the protein 5bnr were obtained from the Protein Data Bank (PDB) database, and the crystal structure was refined to ensure its high quality and reliability for use in molecular docking studies to explore the interactions between the proposed compounds and E. coli Fabh. Table 3 presents the physicochemical attributes of 5bnr, including the cell space group, crystallographic resolution, molecular weight, amino-acid chain name, and number of amino-acid residues. These attributes are important in understanding the structure and properties of the protein and can be used to investigate its function and interactions with other molecules. The crystal structure of 5bnr was refined through a series of steps, including correcting bond orders, formal charges, and missing hydrogen atoms, as well as topologies and incomplete and terminal amide groups. The crystal structure was also minimized using a force field to reorient side-chain hydroxyl groups, and the minimization was restricted to the input protein coordinates by a predefined Root Mean Square Deviation (RMSD) tolerance of 0.3 Å. These steps ensured that the crystal structure is of high quality and can be used reliably in molecular docking studies to explore the interactions between the proposed compounds and E. coli Fabh.
Table 3
The physicochemical attributes of 5bnr have been obtained from the PDB repository.
Protein 5BNR: From Amino-Acid Chains to Physiochemical Properties
|
Parameters
|
Value
|
Cell Space Group
|
P 41 21 2
|
Crystallographic Resolution
|
1.89 Å
|
Molecular Weight
|
33.88 kDa
|
Amino-acid Chain Name
|
A
|
Number of Amino-acid Residues
|
309
|
3.3.3. The Potential Binding Location of E. coli Fabh Protein using MOE and Active Site Analysis
As a result of this, the potential binding location of the E. coli Fabh protein was predicted by utilizing the MOE 2015.10 software after completing the energy minimization process of the protein complex. The software analyzed the filled volume of the active site with the known ligand to determine the protein binding site. To do this, the co-crystallized molecule was carefully selected, and a spherical boundary was generated around it using the "define sphere" option. The radius of this sphere was set at 10 A °, while the X, Y, and Z-axis coordinates were assigned as 14.4, 14.4, and 14.4, respectively, which was chosen from the selection option. Figures 5 and 6 represent the active pocket site residues of the prepared protein. This approach enabled to accurately determine the potential binding location of the E. coli Fabh protein, which is important in understanding its function and interactions with other molecules. The findings presented in this part of the research paper contribute to the growing body of research aimed at identifying new treatments for bacterial infections.
3.3.4. Refined Ligand Preparation for Accurate Compound Characterization using ACD/ChemSketch
As a result, the chemical makeup of various compounds was meticulously crafted using ACD/ChemSketch and stored in the mol file format to provide an insight into the molecular composition of the compounds, aiding researchers in understanding their properties and potential applications. To delve deeper into the nature of these compounds, the saved files were imported into autodocktools for further examination and analysis, with ligand preparation being a crucial step in this process. The ligand preparation was executed with great precision and accuracy, taking into consideration parameters such as the consistency of oxygen and nitrogen atoms' ionization states at physiological pH, the addition and deletion of hydrogen, and the conversion of the compounds into 3D structures using Chem3D 16.0. This level of attention to detail ensured that the ligands were appropriately prepared, and the compounds were accurately characterized, making it easier to predict their potential interactions with other molecules. This process is essential in drug discovery and other related fields, as it helps to identify and optimize potential candidate compounds. The results are shown in Fig. 7, which provides a visual representation of the prepared ligands and their chemical structure. This approach enabled the researchers to gain a deeper understanding of the molecular properties of the compounds and their potential interactions with E. coli Fabh, which is important in the development of new treatments for bacterial infections.
3.3.5. Computational Validation in Digital Modeling
The use of various software tools and algorithms in this research allowed for a detailed analysis of the E. coli Fabh protein and synthesized compounds. The validation of the protein using MOE software ensured the accuracy of the results, and the prediction of the core structures of the ligands using ChemDraw Professional 16.0 software provided insight into the molecular composition of the compounds. The optimization and minimization of the energy of the ligands' 3D structure using Chem3D 16.0 software allowed for a more accurate prediction of potential interactions between the ligands and the target protein. The docking studies were conducted in MOE, with protein-ligand interactions carried out in the same program. The protein was imported as a ligand-free receptor, and the missing bond orders, hybridization, and charges were assigned to it, which was already implemented in the program. The cavity detection algorithm was used to detect potential binding sites, and the docking was performed with eleven predicted ligands. A cavity docking procedure with an energy grid resolution of 0.45 A° was used, taking the default parameters. The algorithm Mol Dock SE was used, which includes a total of one as the best run, a population size of 50, and the Maximum Interactions of 1500. MOE Virtual Docker was used to visualize the output. The scores of active compounds in scheme 1 were summarized, and the fitness scores for each ligand in the 5bnr receptor were presented in table 4 and Fig. 8. When compared with the score of the standard compound, Streptomycin, which is an anti-microbial agent, most of the compounds in scheme 1 (3a-k) showed good inhibition with more binding affinity. When compared to the standard (score − 7.23) and 3f (score − 6.48), which is a potent inhibitor, most of the compounds in the scheme1 showed good inhibition with more binding affinity. The score of the compounds 3a-k was near to the standard, and the compound 3f (score − 6.48) showed the highest score due to the more electrostatic score (-6.97) and the hydrogen binding with the active site of the target protein as shown in Fig. 9. This approach enabled to identify potential candidate compounds for the development of new treatments for bacterial infections, with the synthesized compounds showing good inhibition and binding affinity towards the E. coli Fabh protein.
Table 4: Docking score for compounds 3a-k with E. coli Fabh Protein pdb (id: 5bnr)
3.3.6. Anti-microbial activity.
The results of the sensitivity test are presented in Table 5 and Fig. 10, which show the testing of synthesized compounds against two bacterial strains and one fungus strain by measuring the sizes of the inhibition zones. Table 5 shows the antimicrobial activity of the tested compounds against S. aureus, E. coli, and C. albicans. The inhibition zones are expressed as the mean ± standard deviation (SD) in millimeters (mm). The results show that compound 3f had the largest inhibition zone against both S. aureus (19.33 ± 2.13 mm) and E. coli (20.11 ± 1.11 mm), while compound 3c had the largest inhibition zone against C. albicans (12.31 ± 3.01 mm). Compounds 3b, 3d, 3e, and 3h showed moderate inhibition against S. aureus, with inhibition zones ranging from 3.11 ± 0.02 mm to 10.21 ± 1.10 mm. Compounds 3c, 3i, and 3j showed moderate inhibition against E. coli, with inhibition zones ranging from 11.12 ± 0.21 mm to 20.21 ± 2.01 mm. Compounds 3j and 3i showed moderate inhibition against C. albicans, with inhibition zones of 5.02 ± 2.25 mm and 7.16 ± 0.17 mm, respectively. The negative control (DMSO) and compounds 3a, 3g, and 3k did not show any inhibition against S. aureus, E. coli, or C. albicans. The positive control (streptomycin) showed positive inhibition zones against all three strains, with inhibition zones ranging from 13.90 ± 1.11 mm to 20.01 ± 1.22 mm. Fluconazole showed positive inhibition against C. albicans, with an inhibition zone of 18.31 ± 2.11 mm, but did not show any inhibition against S. aureus or E. coli. Overall, the results of this table show that compound 3f had the most significant antimicrobial activity against both gram-positive and gram-negative bacteria, and moderate activity against C. albicans. The results also highlight the potential of some of the other compounds to act as moderate antimicrobial agents, but further modifications may be required to improve their efficacy. The positive control (streptomycin) showed positive inhibition against all three strains, demonstrating its effectiveness as an antimicrobial agent. The negative control (DMSO) and some of the synthesized compounds did not show any inhibition against the tested strains, indicating their lack of antimicrobial activity. In addition to the evaluation of the antimicrobial activity of the synthesized compounds against S. aureus, E. coli, and C. albicans, sensitivity and minimum inhibitory concentration (MIC) tests were conducted to determine the effectiveness of the compounds against three clinical isolate organisms. The sensitivity test involved measuring the sizes of the inhibition zones of the synthesized compounds against the clinical isolate organisms. The results showed that compound 3f was the most effective compound against all three clinical isolates, with larger inhibition zones observed compared to the other compounds. The other compounds showed variable levels of effectiveness against the clinical isolates, with some showing moderate inhibition and others showing no inhibition as shown in Fig. 11. Moreover, Table 6 shows the results of the minimum inhibitory concentration (MIC) tests of the synthesized compounds against S. aureus, E. coli, and C. albicans. The MIC values are expressed in milligrams per milliliter (mg/mL). The results show that compound 3f had the lowest MIC values against both S. aureus (5.2 mg/mL) and E. coli (3.4 mg/mL), indicating its high efficacy as an antimicrobial agent. Compound 3c also showed moderate MIC values against S. aureus (14.6 mg/mL) and E. coli (21.4 mg/mL). Compounds 3b, 3d, 3i, and 3j showed moderate MIC values against one or more of the tested organisms, indicating their potential as antimicrobial agents. Compounds 3a, 3e, 3g, 3h, and 3k did not show any inhibition against the tested organisms. The results of the MIC tests provide valuable information on the lowest concentration of the synthesized compounds required to inhibit the growth of the tested organisms. The results demonstrate the potential of compound 3f as a lead compound for the development of new antimicrobial agents with high efficacy against both gram-positive and gram-negative bacteria. The moderate MIC values observed for some of the other compounds also highlight their potential as lead compounds for further development. The results of this research contribute to the ongoing efforts to develop new treatments for bacterial and fungal infections, which are a major public health concern worldwide.
Table 5
Anti-microbial activity of the tested compounds. Inhibition zones are being expressed as the mean ± SD (mm). Staphylococcus aureus, E. coli and C. albicans.
Substance code
|
Inhibition zone by mm
|
S. aureus
|
E. coli
|
C. albicans
|
3a
|
NZ
|
NZ
|
NZ
|
3b
|
3.11 ± 0.02
|
7.11 ± 2.12
|
NZ
|
3c
|
12.31 ± 3.01
|
14.21 ± 1.30
|
NZ
|
3d
|
4.31 ± 1.22
|
4.11 ± 0.12
|
NZ
|
3e
|
3.11 ± 0.10
|
NZ
|
NZ
|
3f
|
19.33 ± 2.13
|
20.11 ± 1.11
|
13.13 ± 0.01
|
3g
|
NZ
|
NZ
|
NZ
|
3h
|
10.21 ± 1.10
|
11.12 ± 0.21
|
NZ
|
3i
|
18.11 ± 1.01
|
20.21 ± 2.01
|
7.16 ± 0.17
|
3j
|
8.28 ± 1.30
|
7.22 ± 1.21
|
5.02 ± 2.25
|
3k
|
NZ
|
NZ
|
NZ
|
Positive control (streptomycin)
|
20.01 ± 1.22
|
17.02 ± 0.21
|
13.90 ± 1.11
|
Fluconazole
|
NZ
|
NZ
|
18.31 ± 2.11
|
Negative control (DMSO
|
NZ
|
NZ
|
NZ
|
Table 6
Minimum inhibitory concentration (MIC) test of the synthesized compounds against the three tested clinical isolates.
Substance code
|
Minimum inhibitory concentration (MIC) by mg/mL
|
S. aureus
|
E. coli
|
C. albicans
|
3a
|
NZ
|
NZ
|
NZ
|
3b
|
25.5
|
23.2
|
NZ
|
3c
|
14.6
|
21.4
|
NZ
|
3d
|
33.9
|
43.3
|
NZ
|
3e
|
NZ
|
NZ
|
NZ
|
3f
|
5.2
|
3.4
|
8.3
|
3g
|
NZ
|
NZ
|
NZ
|
3h
|
NZ
|
NZ
|
NZ
|
3i
|
33.1
|
28.9
|
45.5
|
3j
|
43.2
|
45.5
|
42.1
|
3k
|
NZ
|
NZ
|
NZ
|
3.3.7. A Potential Antimicrobial Agent Against S. aureus, E. coli, and C. albicans, as Determined by Resazurin Reduction Assay Method
The antimicrobial activity of the compound 3f was tested against different microorganisms and MIC values determined visually using resazurin dye reduction assay method. The change in color of the dye from blue to pink indicates the viability of the microbial cells. The enzyme oxido-reductases present inside the unicellular fungi and/or bacterial cells converts the resazurin to resorufin which is pink in color. When the dye color remains blue, then it indicates that there is no activity of viable cells. The added test material kills the bacterial and a fungal cell during incubation was determined by the blue or purple color of dye in the respective wells. The pink color formation in the wells, even after treating with the compound 3f and control drug indicates the presence of viable cells. Thus the least dilution in which the color remains blue was taken as the MIC value. The compound was first weighed out and dissolved in a solution of dimethyl sulfoxide (DMSO) at a concentration of 50 milligrams to select the minimum inhibition concentration as in Table7. The compound 3f was tested for each microorganism at 5 µl and observing the effect of compound 3f on the microbial growth are summarized in Table 8. The results revealed that the compound 3f showed better inhibitory activity to microbial growth. The compound 3f was active against the E. coli and S. aureus bacterial and fungal pathogens C. albicans at the concentrations between 10 − 0.5 µl (100- to 0.004 µg/ml) of the compound 3f as shown in Table7. The MIC values of the compound 3f against the E. coli with MIC of 8.35 µg/mL were the better than that of the antibiotic Streptomycin (10.11 µg/mL). Table 8 presents the minimum inhibitory concentration (MIC) values of compound 3f against three different microorganisms: S. aureus, E. coli, and C. albicans, as determined by the resazurin reduction assay method. The MIC values represent the lowest concentration of the compound that was able to inhibit the growth of the microorganisms [Fig. 12]. The MIC values of compound 3f against S. aureus, E. coli, and C. albicans were found to be 7.13 ± 4.01, 8.35 ± 2.11, and 4.16 ± 1.01 mg/mL, respectively. These values indicate that compound 3f has significant inhibitory activity against all three microorganisms tested. The positive control antibiotics, Streptomycin and Fluconazole, were also tested for comparison. The MIC values of Streptomycin against S. aureus, E. coli, and C. albicans were found to be 6.91 ± 0.21, 10.11 ± 0.11, and 5.11 ± 3.01 mg/mL, respectively. The MIC values of Fluconazole were not determined for S. aureus and E. coli, but for C. albicans, the MIC value was found to be 2.21 ± 0.01 mg/mL. The negative control used in the study was dimethyl sulfoxide (DMSO), which showed no inhibitory effect on the growth of the microorganisms. The results demonstrate that compound 3f has significant inhibitory activity against the microbial pathogens S. aureus, E. coli, and C. albicans, with MIC values comparable to or better than those of the positive control antibiotics. The findings suggest that compound 3f has the potential to be developed as a new antimicrobial agent for the treatment of infections caused by these microorganisms.
Table 7
The zones inhibition value by different concentration of the compound 3f on three clinical isolates organisms
Organisms
Concentration µl
|
S.aureus
|
E.coli
|
C. albicans
|
100
|
94.11 ± 0.35
|
87.07 ± 0.13
|
83.07 ± 0.13
|
50
|
7.12 ± 0.01
|
8.11 ± 1.11
|
8.11 ± 02.21
|
25
|
6.81 ± 0.01
|
7.92 ± 0.04
|
7.12 ± 1.20
|
12.5
|
5.91 ± 0.01
|
7.01 ± 0.21
|
6.31 ± 1.11
|
6.3
|
3.91 ± 0.01
|
6.91 ± 0.10
|
5.91 ± 2.33
|
3.1
|
0.63 ± 0.11
|
1.11 ± 0.20
|
2.91 ± 0.11- strp
|
1.6
|
0.61 ± 0.01
|
1.201 ± 1.20
|
1.51 ± 0.04
|
0.8
|
0.31 ± 0.10
|
0.681 ± 0.10
|
1.721 ± 0.01
|
0.4
|
0.051 ± 0.12
|
0.161 ± 1.10
|
1.634 ± 2.11
|
0.2
|
0.0051 ± 0.01
|
0.09 ± 0.01
|
1.12 ± 0.11
|
Strp (5 µl )
|
0
|
0
|
0
|
Flu (5 µl )
|
0
|
0
|
0
|
NC
|
100
|
100
|
100
|
Table 8
Minimum Inhibitory Concentration (MIC) Values of Compound 3f Against S. aureus, E. coli, and C. albicans Determined by Resazurin Reduction Assay Method
comp
|
Minimum inhibitory concentration (MIC) by mg/mL
|
S. aureus
|
E. coli
|
C. albicans
|
3f
|
7.13 ± 4.01
|
8.35 ± 2.11
|
4.16 ± 1.01
|
P.C (Streptomycin)
|
6.91 ± 0.21
|
10.11 ± 0.11
|
5.11 ± 3.01
|
P.C (Fluconazole)
|
NZ
|
NZ
|
2.21 ± 0.01
|
NC (DMSO
|
NZ
|
NZ
|
NZ
|
The blue or purple color of the dye in the wells indicates bacterial or fungal growth inhibition, while the pink color indicates the presence of viable cells. The figure shows that compound 3f has inhibitory activity against all three microorganisms tested, with E. coli and S. aureus showing a significant reduction in growth in the presence of the compound. The figure also demonstrates that compound 3f has a similar inhibitory effect on C. albicans as Fluconazole, a commonly used antifungal drug. Inhibition of biofilm formation by the compound 3f.
3.3.8. The Anti-Biofilm Activity of compound 3f against S. aureus, E. coli, and C. albicans biofilms
The study investigated the anti-biofilm activity of compound 3f against three clinical isolates of S. aureus, E. coli, and C. albicans. Biofilms were formed by inoculating the isolates into biofilm media with and without compound 3f. The results showed that compound 3f effectively inhibited biofilm formation by all three microorganisms. The anti-biofilm activity was demonstrated by comparing the biofilm formation in the clinical isolates with and without treatment of compound 3f. Figure 13 depicts the difference in biofilm formation between the untreated and treated isolates. The untreated biofilms showed dense growth, while the treated biofilms showed a significant reduction in density. Table 9 presents the optical density (OD) values of biofilms formed by three different microorganisms, S. aureus, E. coli, and C. albicans, before and after treatment with compound 3f. Biofilms are communities of microorganisms that adhere to a surface and are protected by a self-produced extracellular matrix. Biofilms can be difficult to treat with antimicrobial agents, as they are often resistant to traditional treatments. The OD values of the untreated biofilms of S. aureus, E. coli, and C. albicans were 0.42, 0.61, and 0.28, respectively. These values indicate the density of the biofilm formed by the microorganisms. After treatment with compound 3f, the OD values decreased significantly, indicating a reduction in biofilm density. The treated biofilms of S. aureus, E. coli, and C. albicans had OD values of 0.19, 0.35, and 0.12, respectively. The results of Table 9 demonstrate that compound 3f has significant activity against biofilms formed by S. aureus, E. coli, and C. albicans. The reduction in OD values indicates a decrease in the density of the biofilm, which can lead to increased susceptibility of the microorganisms to antimicrobial agents. This finding suggests that compound 3f has the potential to be developed as a new antimicrobial agent for the treatment of biofilm-associated infections caused by these microorganisms. Biofilm formation is a common mechanism for microbial survival and can be a significant factor in the development of chronic infections. The ability of compound 3f to inhibit biofilm formation may make it a promising candidate for the treatment of biofilm-associated infections caused by these microorganisms.
Table 9
Optical Density (OD) Values of Biofilms of S. aureus, E. coli, and C. albicans before and after treatment with compound 3f. The table displays the OD values of the untreated biofilms and those treated with compound 3f, indicating a significant reduction in biofilm density after treatment with the compound 3f.
Isolates microbes
|
OD values
|
Untreated-3f
|
Treated-3f
|
S. aureus
|
0.42
|
0.19
|
E. coli
|
0.61
|
0.35
|
C. albicans
|
0.28
|
0.12
|