Abstract
Benzothiazoles, an anticonvulsant, antiviral, antihypertensive, and cancer-fighting medication of the heterocyclic scaffold family, also acts as antibacterial and antiviral agents. There is much interest in this chemical’s production because of the strong and vital biological action it possesses. Substituted aromatic aldehydes were combined with 2-amino-benzothiazole-6-sulfonic acid amides, or Schiff base derivatives, to create Schiff base derivatives. Recrystallized, characterized, and tested for diuretic efficacy in vivo using online tools, m.p. (melting point), Rf, FTIR (Fourier transform infrared), 1H-NMR (proton nuclear magnetic resonance) data The molecular characteristics of all the substances created were estimated using Lipinski’s rule of 5, OSIRIS (software) molecular property explorer, Molsoft, and Autodock 4.0 docking software. Male Wistar rats were used to make all the compounds traditionally in order to test for diuretic activity. Neither the elemental nor the spectral information for the synthesized compounds disagreed. There were five different methods used to evaluate these compounds: Lipinski rule of five, Molsoft to determine molecular characteristics, PASS (prediction of activity spectra for substances) values to determine the diuretic effect, and OSIRIS software to determine toxicology. In order to investigate the diuretic effects of the selected drugs, docking analysis was used. Acetazolamide was shown to have a diuretic effect that was superior to that of compounds IIIb and IIIe, whereas 2-{(E)-[(3-hydroxyphenyl)methylidene]amino}-1,3-benzothiazole-6-sulfonamide (IIIb) was found to be the most promising potential.
1. Introduction
Patients with life-threatening diseases such as congestive heart failure, renal disease, hypertension, acute oedema of the lungs, and ascites may all improve from drug-induced diuresis. Because diuretics all cause potassium loss even if they work well at boosting sodium excretion, researchers have been searching for new diuretic medications that do not cause potassium loss yet still work well at maintaining therapeutic efficacy [1, 2].
Derivatives of 2-aminobenzothiazoles are reported to have diverse biological activities like cytotoxicity, anti-inflammatory, analgesic, anthelmintic, antiviral, antidiabetic, antimicrobial, antileishmanial, anticonvulsant, Alzheimer’s disease, and calcium channel blocking [3–6]. The present study was carried out on 6-sulfonamide containing 2-aminobenzothiazole Schiff bases derivatives as lead molecules of the study with the aid of docking for evaluating their diuretic activity, which is an adjutant therapy in treating hypertension [7–9].
It is a common practice in SBDD to use molecular docking because it can accurately anticipate the shape of small-molecule ligands in the target-binding site. In addition, it is one of the most reliable approaches. Molecular docking has been an essential method in drug discovery since the first algorithms were developed in the 1980s [10]. In order to find the most likely binding configurations, two processes must be completed: A wide range of probable binding modes is examined, and the interaction energy associated with each of these binding conformations is accurately predicted [11].
The software used to determine if a molecule is a drug or not follows the Lipinski rule of five [12]. It includes information on molecular weight, hydrogen bond donor, hydrogen bond acceptor, logP value, and molar refractivity. Molsoft L.L.C drug likeness and molecular property prediction provides a molecule’s molecular formula, molecular weight, hydrogen bond acceptor, hydrogen bond donor, MolLogP, MolLogS, MolPSA, MolVol, pKa, BBB score, number of stereo centres, and drug likeness score [13–15]. OSIRIS property explorer offers information if a molecule [16], if produced, creates any toxicity impact by exhibiting in the window on the screen,; green colour indicates nontoxic while red colour harmful. Other data like solubility, TPSA, clogP, drug likeness, and drug score are also provided. The online freeware PASS (prediction of activity spectra for substances) determines whether a chemical is physiologically active as Pa or inert as Pi. Molinspiration and SWISS ADME [17] software’s were used to determine drug activity ADME, physicochemical characteristics, GI absorption, drug likeness, GPCR ligand, ion channel modulator, kinase inhibitor, nuclear receptor ligand, protease inhibitor and enzyme inhibitor, and bioavailability score. Based on the premise, benzthiazoles (Figure 1) have diuretic action as such, the experiment was planned to synthesize derivatives if they exhibit diuretic activity.
2. Materials and Methods
There were three types of beakers used in this experiment: dry Borosil glass, round bottomed glass, and conical glass. MERCK precoated silica gel plates were utilized for TLC monitoring, and a JASCO UV chamber was employed to see spots in TLC.
Instruments for visual melting range measurements were purchased from Lab India. KBr (potassium bromide) disc on FTIR 8300, and KBr press Shimadzu were used to record the IR spectra of compound IIIb. The 1H-NMR spectra of the synthesized chemical IIIb were acquired on a BRUKER-400 MHz spectrometer using DMSO as the solvent, and the results were analyzed. Digital flame photometer Cisco 381 was used for the estimation of ions.
2.1. Chemistry
The synthetic procedures that were followed in order to get the target compounds IIIa-i were represented in diagrams. The structures of synthesized substances were determined on the basis of elemental analysis and spectrum data collected throughout the process. The preceding starting ingredients for the synthesis of 2-amino-benzothiazole-6-sulfonic acid amide (II), which is a cyclisation process, were para-amino sulphanilamide and ammonium thiocyanate. Compounds IIIa-i were first made by reacting equimolar quantities of (II) with suitable aldehydes in 100% alcohol, which is an imine production reaction followed by a nucleophilic addition reaction. Compounds IIIb-i were then synthesized in the same manner [18].
2.1.1. General Method for the Synthesis of 2-amino-benzothiazole-6-sulfonic acid amide (II)
Everything was combined with glacial acetic acid, which had been precooled to 5°C, and 0.06 mol of sodium hydroxide and 0.06 mol of p-aminosulphanilamide. This mixture was kept in an ice bath and mechanically agitated while bromine (0.02 mol) was slowly introduced from an infusion funnel, maintaining a temperature rise of no more than 4–5°C during the process. The results were stunning. For an additional three hours, the mixture was continually churned in an ice bath at a temperature ranging from 0 to 10 °C. Acid was used to remove the hydrochloride salt from the filter and dry it before dissolving it in hot water. The water was used to wash the solution after it had been neutralized with aq. NH3 solution (25 percent) and filtered. The needed pure chemical was obtained using benzene recrystallization (II) [18–20].
Yellowish brown; m.p. 230°C; Yield 80%.
2.1.2. General Method for the Synthesis of Schiff’s Bases of 2-amino benzothiazole Compounds (IIIa-g)
In 20 mL of ethanol, 6-nitro-2-aminobenzothiazole 2a (0.025 mol) was dissolved and 10 mL of ethanol was added in drops to the substituted aromatic aldehyde (0.030 mol). Twenty-four hours at room temperature were spent stirring the reaction mixture. A mixture of ethyl acetate:chloroform (1 : 2) was then evaporated, and the result was recrystallized from this combination to yield (IIIa-g) [21, 22].
2.2. In Silico Tools
Softwares of Lipinski rule of Five, Molsoft, PASS Online, OSIRIS Property Explorer, were used online [23–25].
AutoDock is a programme for simulating molecular models. Protein ligand docking is a particular strength of this method. Vina is an upgraded version of Auto Dock 4.0 and Auto Dock 4.2. Using Auto Dock 4.0, researchers follow four basic phases in the process, including synthesis of proteins, ligands, and grids [11].
2.3. Molecular Docking Studies
Studies of enzyme-ligand interactions in vivo may be predicted by molecular docking studies. In order to do molecular docking experiments on the 3D structure of CA-II enzyme, we used 64-bit operating system under Windows 10 with an HP computer [Intel core i5 8th generation processor, 12 gigabytes of ram]. The 3D structure of the enzyme CA-II was downloaded from the RCSB-Protein Data Bank (PDB ID: 3BL1).
The enzyme’s structure was improved and verified for any missing atoms, bonds, or connections. Remaining residues were carefully removed, omitting ligands and water molecules. Building the ligand molecules required the use of the builder molecule, which reduced their energy. It created an active site by using grid box tools, and an energy-efficient conformer was selected and its energy was minimized. For 3BL1 compounds, IIIb, IIIe, and standard, the optimum binding modes are shown as illustrated in Figures 2–4.
(a)
(b)
2.4. Effect on Electrolyte Concentration
Flame photometry was used to measure the concentration of Na+ and K+ ions. For flame photometric examination, the major standard solutions of sodium and potassium were produced at a weight concentration of 1000 ppm each.
This method was used to dilute these solutions to the correct concentrations before usage. Distilled water was used to dilute the urine (1–3 ml) to a final volume of 100 ml, and the resulting diluted solution was used to measure the ions Na+ and K+ in the urine.
2.5. Evaluation of Diuretic Activity
Methods of diuretic activity were carried out utilizing Lipschitz’s approach. Institutional Animal Ethics Committee 4/IAEC/VCOP/2019 Central Animal House, Vaagdevi College of Pharmacy, Hanamkonda, Warangal approved the study’s experimental procedure.
An acute investigation of diuretic action was conducted on five groups of male Wister rats (n = 6) whose weight was about 100–120 g that had been acclimatized for 10 days. Each group is housed in a metabolic cage with a wire mesh bottom and a urine collection funnel. The faeces are caught in stainless steel sieves in the funnel, which enable the urine to go through. A regular diet of Altromin pellets and water were given to the rats. The rats were fed and watered for fifteen hours before the experiment in order to measure diuretic activity. Gently compressing the pelvic region and tugging the tails of the animals helped empty their bladders. Individual cages for each animal were put 24 hours prior to the experiment’s start date for environmental adaption. 0.9 percent NaCl solution was used to provide a dosage of 25 and 45 mg/kg of body weight, respectively. Only 0.9 percent NaCl solution is given to the control group. Both 25 and 45 mg/kg body weight of the test compounds were compared to the conventional diuretic acetazolamide in a 0.9 percent NaCl solution (Scheme 1).
A graduated cylinder was used to collect urine, and the volume was recorded every five hours and every 24 hours. Fluorescence photometric analysis was used to determine sodium and potassium concentrations, which were represented as mmol/L [26].
The amount of diuretic activity was determined by using the following formula:
EtOH-Ethanol, GAA-Glacial acetic acid.
3. Results
3.1. In Silico Analysis
The results of Lipinski filtering rule of five, Molsoft’s properties, PASS results are shown in Tables 1–3.
3.2. Docking Analysis
This approach, known as docking, is used to establish the exact binding conformation and orientation of ligand molecules into the active regions of proteins using computer simulation. Nine benzothiazole compounds were synthesized, and the standard (acetazolamide) was docked using AutoDock 4.2.6 against carbonic anhydrase II.
The finest conformers have been identified via the use of the Lamarckian genetic process. During each run of the evolutionary algorithm, the population size was set at 150 persons at random, and the maximum number of energy evaluations was restricted to 2,500,000 for each run. All active site residues in stiff macromolecules were included in the computations since the grid box size was increased to allow for all active site residues. To fit all of the active site residues, the grid box was centered at 9.323 Å × 1.691 Å x 15.842 Å and its X, Y, and Z co-ordinates were all changed to 90, 90, 90 to accommodate the active site residues.
Docking tests demonstrated that the top 5 ligands had the lowest affinity for the target protein carbonic anhydrase II, when compared to all other ligands evaluated in this study. The binding energy (Kcal/mol) and the number of hydrogen bonds formed with active site areas of the protein were measured and analyzed in the study of protein-ligand interactions. With the use of the Chimera 1.13.1 viewer, the docking contacts between the six ligands and carbonic anhydrase protein were seen and are shown in Figures 2–5. The final docked confirmation for the different ligands is shown in Tables 4–7 in accordance with the binding energy, the number of hydrogen bonds formed, the bond distance, and the interacting residues. Third, IIIb and acetazolamide had the lowest binding energy, with a docking score of −5.63 Kcal/mol (creating four hydrogen bonds with HIS) and −3.38 Kcal/mol (forming two hydrogen bonds with each of HIS and ASN), respectively.
3.3. Experimental
Compound IIIb: FTIR (cm−1): 3376 (O-H str), 3154 (aromatic C-H str), 2923 (aliphatic C-H str), (C = N str), 1078 (C-N str), 1459 (C = C str), 1376 (SO2NH2 str), 721 (disubstituted benzene), 829 cm−1 (asymmetrically substituted benzene) (Figure 6).
1H NMR (δ ppm, DMSO): 2.4 (s,1H, C-H); 6.9 (d, 1H,ArH); 7.0–7.4 (m, 3H, ArH); 7.8–8.0 (2d, 2H, ArH); 7.9 (d, 1H, ArH); 8.4 (s, 1H, ArH); 10.0 (s, 2H, NH2) (Figure 7).
3.4. Diuretic Activity
The in vivo diuretic activity of the benzothiazole derivatives was determined using a conventional technique against male Wistar rats, and the results were compared to those of the reference compound (Tables 6–7 and Figures 8–10).
Both the synthesized compounds (IIIb) are found to be equipotent and (IIIe) is more potent than the standard drug (acetazolamide).
4. Discussion
Based on the results of Lipinski rule of five, the compounds synthesized were obeying Lipinski rule of 5. PASS values suggesting that all the synthesized compounds obey the PASS values. The standard (acetazolamide) has action nonselectivelyon all the sub classes of carbonic anhydrase enzyme. The molecules IIIb, IIIc, IIId, IIIe, IIIf, and IIIg have Pa > 0.1 primarily acting on carbonic anhydrase II, but the standard with Pa > 0.3 acting on all the subtypes of the enzyme. Toxicity (tumorigenic and reproductive effects) is present in the normal medicine, but not in the synthesized molecules except for IIId and IIIf, according to the OSIRIS molecular property explorer. The length of the hydrogen bonds produced with the interacting residues in the docking study findings reveals that the bonding was excellent for all of the ligands tested. The PDB sum predicted most of the important residues presented in Table 4 to be active site residues. All of the ligands have docking contacts with the protein carbonic anhydrase II, according to the docking score. This research used ligands having diuretic activity, which may have a distinct mechanism of action. Using molecular docking simulations, this research found that the IIIb and IIIe, block carbonic anhydrase II.
All of the more modern benzothiazole derivatives were created in the laboratory by mixing a number of different chemical reagents together. Hydroxyl derivatives were more productive in this case. TLC and melting points of the synthesized compounds differed significantly from those of the first stage product. One component, IIIb, was purified using column chromatography and characterized using spectroscopic methods including FTIR and 1H NMR after it was produced and recrystallized from ethanol.
5. Conclusion
The results of the evaluation of diuretic activity indicated that the compound IIIb having urinary excretion, diuretic action, diuretic activity, and Lipschitz values higher than the standard drug which indicate that it is more potent than the standard. From Table 7 and Figures 8 and 9, it is evident that the sodium excretion is more and potassium excretion is moderate to that of the standard and urea-treated groups at 45 mg/kg dose.
Data Availability
All data used to establish the conclusions of this study are integrated into the article.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Acknowledgments
The authors extend their appreciation to the Deanship of Scientific Research at King Khalid University (KKU) for funding this work through the Research Group Program Under the Grant Number (R.G.P.1/256/43). This research received support by the Taif University Researchers Supporting Project, number (TURSP-2020/242), Taif University, Taif, Saudi Arabia.