Current Trends in Pharmacy and Pharmaceutical Chemistry

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Current Trends in Pharmacy and Pharmaceutical Chemistry is the official Journal of Ateos Foundation of Science Education and Research, hosted and Managed IP Innovative Publications Pvt. Ltd, New Delhi, India. Current Trends in Pharmacy and Pharmaceutical Chemistry is an open access, peer-reviewed quarterly international journal publishing since 2019 and is published under auspices of the Ateos Foundation of Science Education and Research. It aims to uplift researchers, scholars, academicians, and professionals in all academic and scientific disciplines. more...

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Get Permission Narasimhan, Gokul kumar K, Balalji, Naachiar, Khiren S, and Harikrishnan N: Comprehensive investigation of two substituted benzimidazoles: Design, synthesis, biological assessment, and characterization


Introduction

The main focus of medicinal chemistry is designing and creating pharmaceutical drugs with the desired therapeutic activity and intended use. Benzimidazole belongs to a significant class of biologically active heterocyclic compounds and plays a major role in medicinal chemistry. One of the earliest nitrogen heterocycles known to science. This bicyclic compound consists of the fusion of benzene and imidazole. Benzimidazoles are a group of molecules that showed potential for use in various pharmacological purposes. Since its derivatives have a variety of biological activities, including antioxidant, antimicrobial, anthelmintic, anticancer, antihypertensive, antineoplastic, anti-inflammatory, analgesic, antifungal, and antiviral properties, the chemistry and pharmacology of benzimidazole are of great interest to medicinal chemistry. A large variation of 2-replaced benzimidazole was found to contain anti-inflammatory, antispasmodic, antihistamine, antimicrobial, anti-tumor, and cyclooxygenase inhibitor activity. Various 2-substituted benzimidazole compounds include Mebendazole, Flubendazole, Parbendazole, Albendazole, Pimobendan, and Omeprazole.

Molecular docking is a key tool. Predicting the dominant binding mode of a ligand with a protein having a known three-dimensional structure is the objective of ligand-protein docking.1, 2 For candidate ligands being docked into a protein target with high binding energies, a scoring function is used. The macromolecule serves as the protein receptor in this process. The ligand molecule, which can function as an inhibitor, is a micromolecule. A class of antiparasitic medications known as anthelmintics, or antihelminthics, eliminates internal parasites such as parasitic worms from the body by either killing or stunning them, all without posing a serious risk to the host. They are also called “vermifuges” and “vermicides”. Toxicants, such as anthelmintics, must be specific to the parasite. Certain biological activity is indicated by the presence of multiple phytochemical contents and strong antioxidant activity observed in anthelmentics. Antioxidants are substances that inhibit the production of free radicals through the chemical process of oxidation. The target parasite can affect the effectiveness of benzimidazole anthelmintics; whipworms are less responsive to these drugs than Ascaris nematodes. The β-tubulin isotypes of soil-transmitted helminths and ascarids were identified, and phylogenetic analysis revealed that the ascarids have a similar repertoire of seven β-tubulin isotypes. 3, 4, 5

Figure 1

Scheme of the work

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Materials and Methods

Procedure for synthesis

The process of synthesizing 2-substituted benzimidazole was done by using 1.08 g of ortho-phenylene diamine, which was kept in the Round Bottom Flask (RBF). The contents were weighed accordingly, as follows: 1.38 g salicylic acid, 2.18 g sulfosalicylic acid, 2.12 g 3,5-dinitrobenzoic acid, 0.75 g glycine, and 0.92 g thioglycolic acid. Then, ethanol was added to the RBF. The porcelain chips were also added in small quantities to avoid overheating or bumping. Then the RBF was connected to the reflux condenser with outlet and inlet pipes and a continuous water supply. It was kept in the heating mantle at a constant temperature for 2-3 hours. After the process of heating the compound, the sample was poured into the ice water, and then sodium hydroxide was added drop by drop to obtain the product. The same process was repeated for all the compounds for the synthesis.

Determination of DPPH Method: 4

The product sample of each compound is weighed at 10 mg separately and diluted with ethanol to make 10 ml using a standard flask. The extracted compound was then made in various concentrations. The concentrations were obtained by serial dilution for each compound, ranging from 1000µg/ml, 500µg/ml, 250µg/ml, 125µg/ml, 62.5µg/ml, 31.25µg/ml, 15.625µg/ml, and 7.81µg/ml. Here, we're applying the DPPH method to determine the antioxidant properties of the product. The ascorbic acid is taken as the standard solution with different concentrations of 100µg/ml, 200µg/ml, 300µg/ml, 400µg/ml, and 500µg/ml by serial dilution. Ethanol is taken as the control. A test tube containing 1 ml of the extracted compound and 1 ml of the freshly made DPPH solution was filled with the mixture. The free radical property of the compound was indicated by a colour change from violet to yellow. Then it is determined by using the spectrophotometer and measuring the absorbance at 517 nm. After the absorbance, the percentage inhibition and IC50 values were determined.

Determination of ABTS Method

The phosphate buffer solution with a pH of 6.8 was prepared in a 100-ml volumetric flask. [Preparation of phosphate buffer solution pH-6.8: 0.1 g of KH2PO4, 0.2 g of K2HPO4, and 0.85 g of NaCl were dissolved in distilled water up to 100 ml in the volumetric flask]. Then 0.0662 g of ammonium per sulfate was added to the phosphate buffer solution in the volumetric flask and made up to 100 ml. 0.3841 g of ABTS was weighed and mixed with the above prepared buffer solution up to 10 ml in the volumetric flask, making the ABTS stock solution.

The product sample of each compound is weighed at 10 mg separately and diluted with ethanol to make 10 ml using a standard flask. The extracted compound was then made in various concentrations. The concentrations were obtained by serial dilution for each compound, ranging from 1000µg/ml, 500µg/ml, 250µg/ml, 125µg/ml, 62.5µg/ml, 31.25µg/ml, 15.625µg/ml, and 7.81µg/ml. Here, we're applying the ABTS method to determine the antioxidant properties of the product. The ascorbic acid is taken as the standard solution with different concentrations of 100µg/ml, 200µg/ml, 300µg/ml, 400µg/ml, and 500µg/ml by serial dilution. Ethanol is taken as the control. Various test tubes were taken, each containing 1 ml of product of different concentrations, and 1 ml of ABTS solution was filled with the mixture. The free radical property of ABTS is that the dark blue color disappears when it combines with an antioxidant compound. Then it is determined by using the spectrophotometer and measuring the absorbance at 734 nm. After the absorbance, the percentage inhibition and IC50 values were determined.

In-Silico Screening 2

Ligand prepation

The structures of the product were drawn using the chemsketch software. Then the smile notations were extracted using structure using an online SMILES translator. SMILES notations were converted to PDB format. The file format designated as "PDB" was selected for the output. Three-dimensional coordinates and the SMILES representation "Kekule" were selected. The ligand in PDB file format is downloaded.

Protein preparation

1SA0 TUBULIN-COLCHICINE: STATHMIN-LIKE DOMAIN COMPLEX was downloaded from the protein data bank in the PDB format. PDB DOI: https://doi.org/10.2210/pdb1SA0/pdb

Figure 2

PDB structure of protein ISA0

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Molecular docking

This study deals with the evaluation of the anthelmintics using 2-substitued benzimidazole using in silico docking studies. In-silico docking studies were carried out using PyRx software. Three important parameters, like binding energy, inhibition constant, and intermolecular energy, were determined.

Lipinski rule of 5 analyzer 6

The analysis of the Lipinski Rule of 5 for the product sample was performed using the online website scfbio@iitd.

The PDB format of the chosen product was selected and uploaded.

The pH was set at 7.

The chosen file format was submitted.

After submitting the file, the results table will be provided along with the mass, hydrogen bond donor, hydrogen bond acceptors, log P, and molar refractivity.

Results

Table 1

Theoretical and practical yield of compounds

Compounds

Theoretical Yield

Practical Yield

Melting point

Rf

Salicylic Acid

2.1g

0.042g

320°C

0.12

Sulphosalicylic Acid

2.9g

4.420g

340°C

0.42

Dinitrobenzoic Acid

2.8g

2.901g

280°C

0.6

Thioglycolic Acid

1.64g

2.417g

260°C

0.45

Glycine

1.47g

2.054g

310°C

0.54

Figure 3

ATR of dinitrobenzoic acid derivative

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Figure 4

ATR of glycine derivative

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Figure 5

ATR of salicylic acid derivative

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Figure 6

ATR of sulphosalicylic acid derivative

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Table 2

Analysis of compounds through Lipinski’s Rule of Five

Compound

Molecular weight <500

H-bond donors <5

H-bond acceptors <10

LogP <5

Molar refractivity (40-130)

Number of Lipinski′s rule violation

Salicylic Acid

210.236

2

2

2.936

63.195

0

Sulfosalicylic Acid

290.3

3

4

2.182

73.009

0

Dinitrobenzoic acid

284.231

1

5

3.045

74.839

0

Glycine

147.181

2

2

1.022

43.985

0

Thioglycolic Acid

164.233

2

2

1.993

48.784

0

Table 3

Molecular docking studies of the compounds against the target

Ligand

Binding Affinity

No of hydrogen bond

Interaction

Glycine

5.2

Two hydrogen bond GlU A 411

Pi-Pi stacking Pi-alkyl

Thioglycolic Acid

4.4

Nil

Pi-Pi stacking Pi-alkyl

Dinitrobenzoic Acid

6.2

Nil

Pi-Pi stacking Pi-alkyl

Salicylic Acid

6.4

Nil

Pi-Pi stacking, pi-alkyl and pi-sigma

SulphoSalicylic Acid

6.5

One -ARG A 105

Pi-Pi stacking, pi-alkyl and pi-sigma

Standard Albendazole

5.2

Nil

Pi-Pi stacking, pi-alkyl and pi-sigma, Pi-sulfur

Figure 7

The glycine derivative interact with the target

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Figure 8

The sulphosalicylic acid derivative interaction with target

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Figure 9

The sulphosalicylic acid derivative interaction with target

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Figure 10

The sulphosalicylic acid derivative formed hydrogen bond with the target

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Figure 11

Thioglycolic acid derivative interaction with the target

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Table 4

Antioxidant activity of compounds

S.NO

Compound

IC50 Value Mcg/ml DPPH method

IC50 Value Mcg/ml ABTS method

1

Salicylic Acid

37

3.5

2

Sulphosalicylic Acid

Above 200

Above 200

3

Dinitrobenzoic Acid

190

Above 200

4

Thioglycolic Acid

130

180

5

Glycine

360

Above 150

6

Ascorbic acid

60

1.5

Figure 12

Antioxidant activity of compounds using DPPH Method

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Figure 13

Antioxidant activity of ascorbic acid

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Figure 14

Antioxidant activity of compounds using ABTS method

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Discussion

The dinitrobenzoic acid derivative had a binding affinity of –6.2. The benzimidazole part had pi-pi stacking with the residue TRY A 407. The benzene fused with imidazole formed a pi-alkyl interaction with the residue VAL A 182, and the phenyl part formed a pi-alkyl interaction with the residue ALA A 100. Salicylic acid derivatives had a binding affinity of -6.4. The benzimidazole part had pi-pi stacking with the residue TRY A 407 . The benzene fused with imidazole formed a pi-alkyl interaction with the residue VAL A 182, and the phenyl part formed a pi-alkyl interaction with the residue ALA A 100. The phenyl part of the derivative had a pi-sigma interaction with the residue ALA A 100. The derivative of sulphosalicylic acid exhibited a binding affinity of -6.5 towards the target 1SA0. It shared a single 2.87 A hydrogen bond with residue ARG A 105. There was pi-pi stacking in the benzimidazole portion with residue TRY A 407. Pi-alkyl interactions were established by the benzene fused with imidazole with residue VAL A 182 and the phenyl portion with residue ALA A 100. The derivative's phenyl component interacted pi-sigma with the residue ALA A 100. With target 1SA0, the glycine derivative had a binding affinity of -5.2. Two hydrogen bonds had been created. Two hydrogen bonds were established between the free hydrogens and the residue GLU A 411. It measured 3.31 and 3.36 A in length, respectively.

There was pi-pi stacking in the benzimidazole portion with residue TRY A 407. Pi-alkyl interactions were established by the benzene fused with imidazole with residue VAL A 182 and the phenyl portion with residue ALA A 100.A derivative of thioglycolic acid had a binding affinity of -4.4. There was pi-pi stacking in the benzimidazole portion with residue TRY A 407. Pi-alkyl interactions were established by the benzene fused with imidazole with residue VAL A 182 and the phenyl portion with residue ALA A 100.A derivative of dinitrobenzoicacid had a binding affinity of -6.2. There was pi-pi stacking in the benzimidazole portion with residue TRY A 407.Pi-alkyl interactions were established by the benzene fused with imidazole with residue VAL A 182 and the phenyl portion with residue ALA A 100. The derivative of salicylic acid demonstrated a binding affinity of -6.4. The residue TRY A 407 in the benzimidazole portion displayed pi-pi stacking. Pi-alkyl interactions were established by the benzene fused with imidazole with residue VAL A 182 and the phenyl portion with residue ALA A 100. The derivative's phenyl component interacted pi-sigma with the residue ALA A 100.With a binding affinity of -4.4, the derivative of thioglycolic acid exhibited pi-pi stacking in the benzimidazole portion, containing the residue TRY A 407. Pi-alkyl interactions were established by the benzene fused with imidazole with residue VAL A 182 and the phenyl portion with residue ALA A 100.TRY A 407 was the residue with pi-pi stacking in the benzimidazole portion, while albendazole had a binding affinity of -5.4. Pi-alkyl interactions were established by the benzene fused with imidazole with residue VAL A 182 and the phenyl portion with residue ALA A 100. Pi-sulfur interaction was observed with PHE A 404.Overall, these results highlight the importance of specific molecular interactions, such as pi-pi stacking, hydrogen bonding, and pi-alkyl interactions, in dictating the binding affinities and specificities of the studied derivatives with their target proteins. Understanding these interactions is essential for rational drug design and optimization efforts aimed at developing potent and selective therapeutic agents.

Theoretical and practical yields of compounds provide insight into the efficiency of chemical reactions and the practicality of synthesis processes. In this table, we observe the theoretical and practical yields of five different compounds, along with their melting points and Rf values. The theoretical yield of salicylic acid was 2.1 g, but the practical yield was significantly lower at 0.042 g. This discrepancy suggests issues with the synthesis process or purification methods. The practical yield of sulphosalicylic acid exceeded its theoretical yield, indicating a successful synthesis with a practical yield of 4.420 g compared to a theoretical yield of 2.9 g. The melting point and Rf value align with the expected characteristics of this compound. Both the theoretical and practical yields of dinitrobenzoic acid were close, indicating a relatively efficient synthesis process. The melting point and Rf value also match the expected properties of this compound. The practical yield of thioglycolic acid exceeded its theoretical yield, indicating a successful synthesis process with a practical yield of 2.417 g compared to a theoretical yield of 1.64 g. The melting point and Rf value align with the expected characteristics of this compound. Similar to thioglycolic acid, the practical yield of glycine also exceeded its theoretical yield, indicating a successful synthesis process with a practical yield of 2.054g compared to a theoretical yield of 1.47g. The melting point and Rf value align with the expected properties of this compound.

The interpretation of ATR (attenuated total reflectance) spectra involves analyzing the absorption bands to identify functional groups present in a molecule. Here's an interpretation based on the provided peaks: Peaks observed at 3384.23 cm⁻¹ and 3361.24 cm⁻¹ correspond to the stretching vibrations of N-H bonds. These peaks indicate the presence of amine or amide functional groups in the molecule. The presence of two peaks suggests that there may be different types of N-H bonds present, such as primary, secondary, or tertiary amines, or different types of amide groups. The peak observed at 3183.13 cm⁻¹ corresponds to the stretching vibration of aromatic C-H bonds. This peak indicates the presence of aromatic rings in the molecule. Aromatic rings typically absorb in this region due to the stretching vibrations of the C-H bonds within the ring structure. Overall, based on the ATR interpretation, the molecule likely contains both amine or amide functional groups and aromatic rings. This information can be useful for further structural elucidation and characterization of the compound.

The Lipinski rule, also known as the Rule of Five, is a guideline in drug discovery and medicinal chemistry used to assess the drug-likeness of chemical compounds. It helps predict whether a compound is likely to be orally bioavailable based on its physicochemical properties. The table provided lists several compounds along with their molecular weight, hydrogen bond donors, hydrogen bond acceptors, LogP (octanol-water partition coefficient), molar refractivity, and the number of Lipinski's rule violations. All compounds listed have molecular weights well below the threshold of 500, hydrogen bond donors, and acceptors, which is favorable according to Lipinski's rule. All the compounds listed have LogP values below 5, indicating good membrane permeability. The compounds listed fall within this range of 40-130, further supporting their drug-likeness. This suggests that all the compounds are likely to have good oral bioavailability based on their physicochemical properties.

The table presents the antioxidant activity of various compounds measured using two different methods: DPPH (2,2-diphenyl-1-picrylhydrazyl) and ABTS (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) assays. Antioxidant activity is expressed as the IC50 value, which represents the concentration of the compound required to scavenge 50% of free radicals in the assay. Salicylic acid shows moderate antioxidant activity, with IC50 values of 37 µg/ml in the DPPH method and 3.5 µg/ml in the ABTS method. Its ability to scavenge free radicals indicates its potential as an antioxidant agent. Sulphosalicylic acid exhibits weak antioxidant activity, as indicated by IC50 values above 200 µg/ml in both the DPPH and ABTS methods. This suggests that it may not be effective in scavenging free radicals at the concentrations tested. Dinitrobenzoic acid shows moderate antioxidant activity with an IC50 value of 190 µg/ml in the DPPH method. However, its activity in the ABTS method is not determined due to the IC50 value being above 200 µg/ml. Thioglycolic acid demonstrates moderate to strong antioxidant activity, with IC50 values of 130 µg/ml in the DPPH method and 180 µg/ml in the ABTS method. Its ability to scavenge free radicals indicates its potential as an antioxidant agent. Glycine shows moderate antioxidant activity with IC50 values of 360 µg/ml in the DPPH method and above 150 µg/ml in the ABTS method. While it exhibits some antioxidant activity, it is less potent compared to other compounds tested. Ascorbic acid, known as vitamin C, serves as a positive control and demonstrates strong antioxidant activity, with IC50 values of 60 µg/ml in the DPPH method and 1.5 µg/ml in the ABTS method. Its potent scavenging ability confirms its well-established role as an antioxidant.

Conclusion

The comparison between theoretical and practical yields provides valuable information about the efficiency and success of chemical synthesis processes. Discrepancies between theoretical and practical yields, as seen in the case of salicylic acid, suggest potential issues with the synthesis or purification methods that need to be addressed. On the other hand, compounds like sulphosalicylic acid, dinitrobenzoic acid, thioglycolic acid, and glycine demonstrated successful synthesis processes, with practical yields either matching or exceeding their theoretical values. This indicates the effectiveness of the synthesis methods used for these compounds. Overall, the combination of theoretical and practical yield data, along with melting points and Rf values, offers valuable insights into the synthesis and characterization of chemical compounds, aiding in process optimization and quality control efforts in chemical research and manufacturing. The ATR spectra confirmed the compounds' functional groups.

The compounds listed in the table adhere to Lipinski's rule, indicating favorable drug-like properties such as appropriate molecular weight, hydrogen bond donors and acceptors, LogP values, and molar refractivity. These properties suggest that the compounds have the potential to be orally bioavailable and may be suitable candidates for further drug development or medicinal chemistry studies. Overall, adherence to Lipinski's rule enhances the likelihood of successful drug discovery and development by identifying compounds with favorable pharmacokinetic properties.

The antioxidant activity of the compounds varies, with some showing moderate to strong scavenging abilities against free radicals. Salicylic acid and thioglycolic acid exhibit moderate antioxidant activity, while ascorbic acid demonstrates strong antioxidant activity, as expected. However, sulphosalicylic acid and glycine show weak to moderate antioxidant activity, indicating their limited efficacy as antioxidants. Further studies could explore the mechanisms underlying the antioxidant properties of these compounds and their potential applications in therapeutic or nutraceutical formulations. Additionally, structure-activity relationship studies may help identify structural features that contribute to enhanced antioxidant activity, aiding in the design of novel antioxidant agents.

Source of Funding

None.

Conflict of Interest

None.

Acknowledgement

Authors wish to acknowledge the Management, Faculty of Pharmacy, DR. MGR Educational and Research Institute, Chennai-77 for providing the necessary facilities required to carry out the research work.

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Article type

Original Article


Article page

106-113


Authors Details

Gopinathan Narasimhan, Gokul kumar K, B Balalji, Azhagu Naachiar, Khiren S, Harikrishnan N


Article History

Received : 13-05-2024

Accepted :


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