INTRODUCTION:

In the Present time, Hypertension has become serious problem. The World Health Organization has reported an increase in the number of patients suffering from this disease. Currently, existing treatments for high blood pressure are not very effective and are generally uncomfortable for patients.¹⁰ This relies in that the patient need to have a very strict control in the dosage and in the moment of the administration of the drug.

Over the last few decades, computational studies together with rational drug design have become a critical part in the development of new drugs. A main target in treatment of hypertension is ACE angiotensin converting enzyme responsible for producing angiotensin II potent vasoconstrictor.¹¹ Therefore, we demonstrated that the proposed heterocyclic compounds are capable of inhibiting ACE activity.

Molecular docking is a key tool in structural molecular biology and computer-assisted drug design.

The goal of ligand–protein docking is to predict the predominant binding mode (s) of a ligand with a protein of known three-dimensional structure. Molecular docking involves computationally exploring a search space that is defined by the molecular representation used by the method, and ranking candidate solutions to determine the best binding mode.

Diphenyl Methanol is an organic compound which consist of 2 phenyl ring. It is widely used as intermediate compound for the synthesis of various main compound.

MATERIAL AND METHODS:

All chemical substances consisting starter materials, reagents and solvents were purchased from the commercial supplier Merck and Sigma-Aldrich. Melting point of the synthesized compounds was done by theil tube capillary method. Purification of the compounds was checked by column chromatography by using Ethyl acetate: Petroleum Ether (2:1). Spots were seen under iodine vapours. IR spectra were obtained from SAIF,Punjab University, Chandigarh.

¹HNMR- spectra were recorded on Bruker Avance II 400 MHz FTNMR Spectrophotometer in CDCl₃ using TMS as the internal standard. (Chemical shift in δ ppm) from SAIF, Punjab University, Chandigarh.

Benzophenone (364 mg, 2.0 mmol) was dissoloved in 25 ml of ethanol. Solution was stirred continuously. Sodium boro-hydride (84 mg,2.2 mmol) solution was added dropwise to benzophenone solution. The progress of reaction was monitered through TLC by using mobile phase Petroleum ether: ethyl acetate (9:1;v:v). Spots on the slides were visualized in iodine chamber. Work up was done by pouring the solution in an ice-bath and adds 4M Hydrochloric acid (3ml). After few minutes, precipitated product was obtained by suction filtration and washed with cold water several times and dried.

Fig: 1

Docking Studies:

(i) Protein preparation:

The protein PDB I.D. (1UZE) is prepared for minimisation by removing water molecules, ligands, hetero atoms and then minimised by applying force field. Active site of protein is identified by using online tools.

(ii) Ligand preparation:

The drug Diphenyl methanol was modified according to the SAR properties and they are saved in. MOL format.

Molecular docking studies was performed by using Glide docking program, Schrodinger Maestro software (version 11.1.012; Schrodinger LLC, New York), held at CDRI Lucknow. The pdb file of Human Angiotensin converting Enzyme (ACE) in complex with Enalapril, was downloaded from protein data bank with pdb id 1UZE.Therefore all these interactions were compared to those reported in the PDB for ACE interactions with the 1UZE.

Fig: 2

Fig:3 Docked Poses of Active site of Diphenyl methanol (PDB Code: 1UZE)

Characterization:

· Solubility

· Melting point

· T.L.C.

· I.R. spectroscopy

· H¹ N.M.R. spectroscopy

Compound name	Diphenyl-methanol
M.F.	C₁₃H₁₂O
M.P.	64-66 °C
Colour	White
Solubility	Soluble in ether and chloroform Slightly soluble in water
Rf value	0.87 [ Petroleum ether: ethyl acetate (9:1;v:v)]
Structure

The progress of reaction was monitered through TLC by using mobile phase Petroleum ether: ethyl acetate (9:1;v:v). Spots on the slides were visualized in iodine chamber & R_f was 0.87

Fig:6 Fig: 7

Pharmacological Activity:

In vitro ACE inhibition assay:

A colorimetric method was used to determine the in vitro ACE inhibitory activity of the synthesized derivatives. The determination began with the preparation of a suspension of 1g rabbit lung acetone powder (Sigma-Aldrich) in 10mL of 0.05M sodium borate buffer (pH 8.2) containing 0.3 M NaCl and 0.5% Triton X-100. This suspension was then centrifuged at 15,000 rpm for 60 minutes at 4°C. The resulting supernatant served as the source of ACE. This assay was based on determination of the level of hippuric acid generated after hydrolysis of the substrate, hippuryl histidyl leucine (HHL; Sigma-Aldrich); the level of hydrolysis was directly related to the inhibitory ability of the compound. A 1mM solution of both test and standard drug (lisinopril) was preincubated with 7mL ACE for 10 minutes at 37°C. To obtain a similar concentration for avoiding the interference, the final volume was adjusted using 0.05 M sodium borate buffer (pH 8.2) containing 0.3 M NaCl. A total of 50mL of 5mM substrate (HHL) was used to start the enzyme reaction, and the mixture was incubated at 37°C. After 30 minutes, the reaction was stopped by adding 0.1mL of 1M HCl. The hippuric acid thus produced was then allowed to react with 0.2mL of pyridine and 0.1mL of benzene sulfonyl chloride (Sigma-Aldrich), to form a yellow solution. ACE inhibition was expressed as percentage inhibition.¹³

Vasodilator activity:

For determination of the vasodilatory effect, Wistar albino rats of either sex, weighing 175–200g, were acquired. The experiment was approved by the Institutional Animal Ethical Committee. The rats were fed a standard laboratory diet and had ad libitum access to water. The hearts of the experimental rats were excised and rapidly transferred to Krebs–Henseleit buffer solution at 37°C. The pericardial and lung tissues were removed and the aorta was cut just below the point of division. After insertion of a glass cannula, perfusion with oxygenated Krebs–Henseleit buffer solution was started. Each heart was then transferred to a glass perfusion apparatus and the heart rate was measured through a chronometer connected to a physiograph. The solution of test compounds (10μg) along with a standard solution (10μg sodium nitroprusside) was administered by injection into the perfusion medium just above the aortic cannula, and the cardiac output was measured using a drop counter. Before recording the parameters induced by BaCl₂, normal heart activity was recorded.¹⁴

RESULT AND DISCUSSION:

The progress of reaction was confirmed by TLC using microscopic slides coated with Silica gel G slurry by pouring method. Spots on the slides were detected in iodine chamber. Melting points of the synthesized compounds were taken by Melting point apparatus. The structures of newly synthesized compounds was established on the basis of IR, ¹H NMR spectroscopy. IC50 of ACE Inhibitors of designed compound was found 4.45 µM. The title compound was prepared according to the general procedure; Rf = 0.86; white solid (yield 2.9g,). ¹H NMR (300 MHz, CDCl₃); δ 7.2 [m], 2 H (Aromatic); CDCl₃, δ 7.5 [m], 8 H (Aromatic); CDCl₃, δ 5.8 [s];1 H (OH); CDCl₃ Calculated for C₁₃H₁₂O. C 60.20%; H 17.19%; O 18.32%. Compound is not showing any kind of hydrogen bonding interaction, may be the probable reason for its lowest activity.

CONCLUSION:

Although the search of curative treatments for Hypertension has long been and is still a priority, improvement of currently prescribed symptomatic treatments remains an important field of research. It can act as an important tool for medicinal chemists to develop newer compounds possessing this moiety that could be better agents in terms of efficacy and safety.

ACKNOWLEDGEMENT:

The author is thankful to School of pharmacy, I.T.M. University Gwalior for providing the facilities to carry out the Research.

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Received on 06.06.2019 Modified on 04.07.2019

Research J. Pharm. and Tech. 2020; 13(1): 101-105.

DOI: 10.5958/0974-360X.2020.00020.7