Molecular docking, synthesis, α-amylase inhibition, urease inhibition and antioxidant evaluation of 4-hydroxy-3-methoxy benzoic acid derivatives

 

Anil Malik, Neelam Malik, Priyanka Dhiman, Anurag Khatkar, Saloni Kakkar*

¹Faculty of Pharmaceutical Sciences, Maharshi Dayanand University, Rohtak-124001, India

 

ABSTRACT:

Different derivatives of 4-hydroxy-3-methoxy benzoic acid were synthesized and evaluated for their antioxidant, α-amylase inhibition and urease inhibition ability. Antioxidant evaluation was performed by DPPH radical scavenging assay and the results revealed that compounds 8, 10 and 13 as most active antioxidant agent with IC₅₀ values of 43.09 µg/ml, 44.59 µg/ml and 43.43 µg/ml respectively. α-Amylase inhibition study was performed using diastase by colorimetric method. Compound 9 showed maximum inhibition with IC₅₀ value of 33.26 µg/ml. Compound 4 was found to possess maximum urease inhibition ability with IC₅₀ value of 35.82 µg/ml. Molecular docking study was performed using autodock software.

 

INTRODUCTION:

Reactive oxygen species (ROS) are the major factors for aging and death of cells¹. In the body, they may generate from several metabolic processes, ultraviolet radiation and other biochemical reactions². These ROS and free radicals produced from oxidative stress may cause degradation of DNA, lipids, proteins and carbohydrates and cause many serious diseases like cancer and diabetes³. Antioxidants have the capability of scavenging various ROS species like hydroxyl radicals (OH), superoxide anion radicals (O₂^-) and hydrogen peroxide (H₂O₂) and thus prevent cells from oxidative degradation⁴.

 

Antioxidants obtained from natural sources like fruits, vegetables and other dietary sources play a significant role in reducing oxidative stress by scavenging free radicals⁵ and keep balance among antioxidants and oxidants⁶. These compounds show antioxidant activity by terminating the chain reaction in lipid oxidation⁷ and chelating metal ions⁸.

 

Diabetes Mellitus is very serious disease of endocrine gland in which there is a disturbance in the metabolism of carbohydrates and lipids⁹. Diabetes affects many organs in the body.  Retardation in hepatic glycogen level due to increase in glycogen catabolism results in hepatic damage¹⁰.  Diabetes is classified into two types namely insulin dependent and non-insulin dependent diabetes in which 10% of type 1 and 90% of type 2 diabetes mellitus cases are reported¹¹.

 

The activity to inhibit the carbohydrate-hydrolysing enzymes like α-amylase is becoming an effective technique to treat type 2 diabetes by blocking the glucose absorption¹². α-Amylase inhibitors retard the rate of starch breakdown and further postprandial blood glucose levels. α-Amylase was the firstly found enzyme¹³. α-Amylase occurs in saliva, pancreatic juice and is a protein enzyme. It breaks alpha bonds of polysaccharides like glycogen, starch and hydrolyze them into glucose and maltose¹⁴. Synthetic antidiabetic drugs have various side effects and this leads to discover newly safe and natural drugs¹⁵. Phenolic compounds among natural sources are found to possess good α-amylase and α-glucosidase inhibitory activity¹⁶.

Urease is a nickel containing enzymatic protein. It is also the first crystallized enzyme¹⁷. Urease is a widely distributed in water, soil and human body¹⁸. Sources of urease are plants, bacteria¹⁹, fungi²⁰ and some invertebrates²¹. Urease consists of two polypeptides namely 21 kDa and 65kDa²². In human body it helps in hydrolysis of urea¹.

 

Urease in high amount is harmful for human tissues and may cause or elevate several diseases like rheumatoid arthritis or atherosclerosis²³.  It is found in human sera as an immunogenic protein and acts as antibodies. Urease is also required by various pathogenic bacteria for maintaining their cells in tissues. Ureolytic activity is responsible for various bacterial infections²⁴. Urease is also used for diagnostic purposes. Ureolytic activity is used to diagnose the Healicobacter pylori infection. Bacterial urease causes many biological disorders like gastritis, peptic ulcers²⁵, kidney stones²⁶ and also cardiovascular disease²⁷.

 

Plants have been used as a medicinal source from very long time to cure diseases of not only humans but also of animals²⁸. Phenolic acids are the rare among natural compounds found in plants and occurs in the form of esters and glycosides²⁹ and possess numerous biological activities like antioxidant³⁰ and anticancer activities³¹. Among phenolic acids reported vanillic acid is a compound of great interest as it possesses numerous biological activities like antioxidant³², antimicrobial³³, anti-inflammatory³⁴, anticancer³⁵, antidiabetic³⁶ and antinoceceptive activities³⁷.

 

From very long time, methods used for the discovery of new drugs like high-throughput screening (HTS) are very long and complicated methods. Currently such methods are replaced by latest and fast methods like virtual screening (VS). VS is the screening of lead compounds by means of computational tools and basically involves two approaches namely ligand-based approach and receptor based approach (molecular docking). Molecular docking generally is a computational method of evaluating perfect match among a ligand and target and involves mainly three steps: recognition of ligand and target, formation of complex (sampling) and identifying the fitness of complex (scoring)³⁸.

 

MOLECULAR DOCKING STUDY:

Molecular docking study is a computational method that describes the best-fit orientation between ligand and protein. Docking mainly involves the association of molecules mainly by hydrogen bonds, pi-pi stacking, hydrophobic interactions, side chain hydrogen bonds, polar interactions etc. First step of docking is to take 3D structure of enzyme. Second step involves the preparation of PDBQT files, grid and docking parameter files by using autodock 4.0. Best poses for a ligand are generated and scored using AutoDock scoring function. Results are analysed by chimera software and biding interactions can be visualised by using pymol software³⁹.

Molecular docking study of urease inhibition:

Automated docking studies were performed using the Auto-Dock version 4.0 as implemented through the graphical user interface AutoDock Tools (ADT 1.5.2). For docking of derivatives, a crystallographic structure having resolution of 2.05Å of Jack Bran Urease (pdb code 3LA4) was used⁴⁰. The standard 3D structures of 4-hydroxy-3-methoxy-benzoic acid derivatives were drawn by ChemSketch drawing package. Polar hydrogen and rotatable bonds were added by Openbabel and Autodock tools, respectively. Current study involves the non-standard protein residues and metal ions for binding site specifications. Some necessary steps taken into consideration were addition of all hydrogen atoms, removal of water molecules from the protein, calculation of Gasteiger charges and merging of non-polar hydrogen atoms to carbon atoms. Ni initial parameters were set as q = +2.0, r =1.170 Å and van der Waals well depth of 0.100 kcal mol^-1. Cubic grid box having 60 Å size (x, y, z) with 0.5 Å spacing and grid maps were made. Grid centre was adjusted to the average coordinates of the two Ni²⁺ ions. For search algorithm, Lamarckian genetic algorithm (LGA) was taken. Lamarckian job involved 10 runs. Settings were adjusted as initial population of 50 structures and maximum number of 2.5 × 10⁶ energy evaluations and generations. Values for other parameters were set as default. This procedure was followed for all synthesized compounds.Those results which differed in positional root-mean-square deviation (RMSD) by less than0.5 Å were gathered together and resultant structures having most appropriate binding energy were selected. Further, for redefining of docking poses, software namely pymol molecular visualization program pymol academic version was used⁴¹.

 

Molecular docking study of α-amylase inhibition:

Three-dimensional structure of α-amylase was taken from protein data bank (1HNY)⁴².The standard 3D structures of 4-hydroxy-3-methoxy-benzoic acid derivatives were drawn by ChemSketch drawing package and energy was minimized using MM2 calculation with a conjugate gradient. The crystal structure of enzyme became free from water molecules by before docking. Auto-dock 4.0 package was used for the molecular docking studies of the inhibitory ligand at the α-amylase binding site with the presence of cofactors (chloride and calcium ions). Active site was determined by using the binding site tools. The docking runs were performed with coordinates x: 11.563, y: 46.792, z: 44.400 and radius of 9 A⁰. Best poses for the derivatives were achieved.

 

MATERIALS AND METHODS:

All solvents and reagents used in this study were procured locally and were of analytical grade. Determination of melting points was done using open capillary tubes on a Sonar melting point apparatus and were uncorrected. Reaction progress was checked by thin layer chromatography (TLC). Products obtained after reaction completion were recrystallized and their purity was analyzed by TLC using silica gel G coated glass plates. ¹H nuclear magnetic resonance (¹H NMR) spectroscopy of all the derivatives was done on Agilent NMR 300 MHz spectrometer and CDCl₃ was used as a solvent. Infra-red (IR) spectroscopy was done on Perkin Elmer Spectrum Two spectrophotometer.

 

General procedure for synthesis of 4-hydroxy-3-methoxy benzoyl chloride (2) (Scheme 1):

Thionyl chloride (0.6 mol) was gently added to vanillic acid (1) (0.35 mol) in a round bottom flask. After addition of thionyl chloride, the reaction mixture was stirred for 4 hours by magnetic stirring. After stirring, mixture was heated to 80⁰ C for 1.5 hours in a water bath. Reaction end point was determined using thin layer chromatography. Solid mass obtained as intermediate was separated out by filtration and further washed with toluene to get 4-hydroxy-3-methoxy-benzoyl chloride (2).

 

General procedure for synthesis of anilides (1-14) (Scheme 1):

To the intermediate (2) (0.025 mol) obtained after first step, solution of different anilines (0.025 mol) (reported in Table 1) prepared in ether was added drop by drop. After addition, the reaction mixture was stirred for 45 minutes to obtain the anilide as precipitate. Temperature of the reaction was maintained at 0-10⁰ C. Anilide was separated out and recrystallized with alcohol.

 

In vitro evaluation of antioxidant activity:

Evaluation of free radical scavenging ability of antioxidant compound was done by DPPH assay and is considered as one of the novel method for antioxidant profile determination. DPPH assay is based upon the reaction of accepting a hydrogen atom by the DPPH from the scavenger molecule i.e. antioxidant, which shows change in deep violet to yellow colour along with contemporary decrease in the absorbance. Change in colour was observed spectrophotometrically and further considered for determining the parameters of derivatives for their antioxidant activity. Vanillic acid derivatives and standard drug were checked for their antioxidant profile by free radical scavenging action of 1,1-diphenyl-2-picrylhydrazyl (DPPH)-free radical activity as given below⁴³.

 

Stock solution preparation:

10 mg of each synthesized compound and standard (ascorbic acid) were weighed properly and taken each into 10 ml volumetric flask. Solution of each compound was made by dissolving them into methanol and volume was maintained to 10 ml. Stock solution concentration was 1000 µg/ ml.

 

Preparation of dilutions:

From the above stock solution, 4 dilutions of each sample and standard were prepared. From the stock solution, 0.25 ml, 0.50 ml, 0.75 ml and 1.0 ml of the sample and standard were pipetted out and transferred to separate 10 volumetric flasks and the volume of each was made up to 10 ml with methanol, which results in the dilutions of sample and standard drug of 25 µg/ml, 50 µg/ml, 75 µg/ml and 100 µg/ml concentrations respectively.

 

Preparation of DPPH solution:

The DPPH solution of 3 µg/ml concentration was made by taking 3.9432 mg of DPPH in 100 ml of methanol into 100 ml volumetric flask.

 

Screening of antioxidant activity:

1 ml of DPPH solution was mixed separately with 1ml solution of each sample and standard drug from each concentration. Control solution was made by adding 1 ml methanol into 1 ml of DPPH solution. All these prepared solutions were placed away from light for 30 minutes and their absorbance was measured at 517 nm. The calculation of percentage inhibition was done by using the following formula⁴⁴.

 

                          A_control - A_sample

% Inhibition = ------------------------X 100

                                A_sample

Where,   A_control = control absorbance

              A_sample = sample absorbance

 

In vitro evaluation of α-amylase inhibitory activity:

Evaluation of α-amylase inhibition activity of each vanillic acid derivative was performed using diastase based on colorimetric method⁴⁵. Soluble potato starch (0.25g) was added in 50 ml of 20 mM phosphate buffer by heating for 15 minutes. Enzyme solution was prepared by dissolving 1 mg diastase into 100 ml of 20 mM phosphate buffer solution (pH 6.9). To prepare color reagent, 5.31 M sodium potassium tartrate with 20 ml of 96 mM 3, 5-dinitrosalicylic acid was dissolved in 8 ml of 2 M sodium hydroxide and 12 ml deionized water. All the vanillic acid derivatives were dissolved in DMSO to give different concentrations.

1 ml of vanillic acid derivative was dissolved in 1 ml solution of enzyme and incubated for 10 minutes at 25⁰ C. In the above solution, 1 ml of color reagent was mixed and test tube was closed. Further, test tube was kept in water bath for 15 minutes at 85⁰ C. The solution was cooled and 9 ml of distill water was added to it. Absorbance of the solution was taken at 540 nm in UV spectrophotometer. Blank solution was prepared by replacing the enzyme solution with buffer solution and absorbance was taken. Control solution was prepared by replacing vanillic acid derivative solution from mixture. Acarbose was taken as standard drug⁴⁶

 

In vitro evaluation of urease inhibitory activity:

Evaluation of urease inhibition activity was done by the method reported by the method reported by Tanaka et al., 2003. DMSO/H₂O mixture (1:1 v/v) was used to prepare stock solutions of vanillic acid derivatives with different concentrations. 250 µL of derivative solution was mixed with 250 µL of jack bean urease (4U) solution. This mixture was pre-incubated at 37⁰ C for 1 hour. After that, 2 ml of 100 mM phosphate buffer (pH 6.8) containing 500 mM urea and 0.002% phenol red as an indicator were added and incubated at room temperature. After incubation, absorbance was measured by UV visible spectrophotometer at 570 nm. Urease enzyme increase the pH of phosphate buffer from 6.80 to 7.7 by producing ammonium carbonate from urea and the end point was determined by the color of phenol red indicator^47,48.

 

RESULT AND DISCUSSION:

Chemistry:

All the vanillic acid derivatives were synthesized in two steps as illustrated in scheme 1. In first step, 4-hydroxy-3-methoxy benzoyl chloride was synthesized. In second step, compounds 1-14 were synthesized by the reaction of anilines with 4-hydroxy-3-methoxy benzoyl chloride (2). The physicochemical properties of all synthesized compounds are shown in Table1.

 

 

Scheme 1: For the synthesis of 4-hydroxy-3-methoxy benzoic acid derivatives

 

Table 1: Arrangement of substituents used for targeted compounds

Compound	R	Compound	R
C-1		C-8
C-2		C-9
C-3		C-10
C-4		C-11
C-5		C-12
C-6		C-13
C-7		C-14

 

Physicochemical and spectral parameters of synthesized compounds:

Characterization of all the synthesized compounds was done by determination of their physicochemical parameters like melting points, R_f value, % yield, FTIR and¹H-NMR spectral studies. The FTIR and ¹H-NMR spectral data was found in agreement with assigned molecular structures. Physicochemical characteristics of synthesized compounds have been represented in Table 2.

 

Table 2: Physiochemical properties of synthesized derivatives (1-14):

^* TLC Mobile phase- Ethyl acetate:Hexane (7:3)

 

Spectral data of synthesized compounds (1-14):

4-hydroxy-3-methoxy benzoyl chloride (intermediate)

IR (KBr pellets)cm^-1: 3479 (O-H str., Ar-OH), 2970 (C-H str., Ar-CH), 1515 (C=C skeletal str., phenyl), 733 (C- Cl str., aromatic).

 

N-(2,6-dimethyl)-4-hydroxy-3-methoxybenzamide (C-1)

IR (KBr pellets) cm^-1: 3419 (O-H str., Ar-OH and N-H str., Ar-NH),3056 (C-H str., Ar-CH), 2941 (C-H str., ether), 2831 (Ar-CH₃), 1601 (C=O str. and N-H bending, 2˚ amide), 1511 (C=C skeletal str., phenyl).

 

N-(3-chlorophenyl)-4-hydroxy-3-methoxy benzamide (C-2)

IR (KBr pellets) cm^-1: 3428 (O-H str., Ar-OH), 3378 (N-H str., Ar-NH), 3065 (C-H str., Ar-CH), 2939 (C-H str., ether), 1600 (C=O str. and N-H bending, 2˚ amide), 1511 (C=C skeletal str., phenyl), 749 (C-Cl str., aromatic),¹H NMR (CDCl₃, δppm): 6.99-7.69 (m ,7H, Ar-H), 7.84 (s, 1H, NH), 3.88 (s, 3H, OCH₃).

 

N-(4-chlorophenyl)-4-hydroxy-3-methoxy benzamide (C-3)

 IR (KBr pellets) cm^-1: 3434 (O-H str., Ar-OH and N-H str., Ar-NH), 3052 (C-H str., Ar-CH), 2940 (C-H str., ether), 1600 (C=O str. and N-H bending, 2˚ amide), 1509 (C=C skeletal str., phenyl), 748 (C-Cl str., aromatic).

 

4-hydroxy-3-methoxy-N-(3-nitrophenyl) benzamide (C-4)

IR (KBr pellets)cm^-1: 3390 (N-H str., Ar-NH and O-H str., Ar-OH), 2948 (C-H str., ether), 1600 (C=O str. and N-H bending, 2˚ amide), 1517 (NO₂ sym. str., Ar-NO₂). ¹H NMR (CDCl₃, δppm): 6.80-8.12 (m ,7H, Ar-H), 8.12 (s, 1H, NH), 3.73(s, 3H, OCH₃).

 

4-hydroxy-3-methoxy-N-(2-nitrophenyl) benzamide (C-5)

IR (KBr pellets) cm^-1: 3479 (O-H str., Ar-OH), 3371 (N-H str., Ar-NH), 3064(C-H str., Ar-CH), 2948 (C-H str., ether), 1605 (C=O str. and N-H bending, 2˚ amide), 1507 (NO₂ sym. str., Ar-NO₂).

N-(2-chloro-4-nitrophenyl)-4-hydroxy-3-methoxy banzamide (C-6)

IR (KBr pellets) cm^-1:3478 (O-H str., Ar-OH),3375 (N-H str., Ar-NH), 2949 (C-H str., ether), 1601 (C=O str. and N-H bending, 2˚ amide), 1505 (NO₂ sym. str., Ar-NO₂), 749 (C-Cl str., aromatic),1H NMR (CDCl₃, δppm): 6.76-8.2 (m ,5H, Ar-H), 8.01 (s, 1H, NH), 4.7 (s, H, Ar-OH).

 

4-hydroxy-3-methoxy-N-(2-methoxyphenyl) benzamide (C-7)

 IR (KBr pellets) cm^-1: 3443 (O-H str., Ar-OH),3377 (N-H str., Ar-NH), 3070 (C-H str., Ar-CH), 2941 (C-H str., ether), 1604 (C=O str. and N-H bending, 2˚ amide), 1509 (C=C skeletal str., phenyl), 1284 (C-O-C str., Ar-OCH3);

 

 

N-(2-chlorophenyl)-4-hydroxy-3-methoxy benzamide (C-8)

IR (KBr pellets) cm^-1:3423 (O-H str., Ar-OH and N-H str., Ar-NH),3061 (C-H str., Ar-CH), 2944 (C-H str., ether), 1600 (C=O str. and N-H bending, 2˚ amide), 1510 (C=C skeletal str., phenyl), 750 (C-Cl str., aromatic), ¹H NMR (CDCl₃, δppm): 6.79-7.51 (m ,7H, Ar-H), 8.12 (s, 1H, NH), 3.89 (s, 3H, OCH₃).

 

4-hydroxy-3-methoxy-N-(3-methoxyphenyl) benzamide (C-9)

IR (KBr pellets) cm^-1: 3443 (O-H str., Ar-OH and N-H str., Ar-NH), 2943 (C-H str., ether) 1602 (C=O str. and N-H bending, 2˚ amide), 1510 (C=C skeletal str., phenyl), 1287 (C-O-C str., Ar-OCH3).

 

N-(2,4-dimethylphenyl)-4-hydroxy-3-methoxy benzamide (C-10)

IR (KBr pellets) cm^-1: 3433 (O-H str., Ar-OH and N-H str., Ar-NH), 3069 (C-H str., Ar-CH), 2941 (C-H str., ether), 1600 (C=O str. and N-H bending, 2˚ amide), 1516 (C=C skeletal str., phenyl), 2835 (Ar-CH₃).

 

N-(4-chloro-2nitrophenyl)-4-hydroxy-3methoxy bezamide (C-11)

IR (KBr pellets) cm^-1: 3473 (O-H str., Ar-OH), 3358 (N-H str., Ar-NH), 2941 (C-H str., ether), 1602 (C=O str. and N-H bending, 2˚ amide), 1507 (C=C skeletal str., phenyl), 746 (C-Cl str., aromatic).

 

4-hydroxy-3-methoxy-N-(4-methoxyphenyl) benzamide(C-12)

IR (KBr pellets) cm^-1: 3428 (O-H str., Ar-OH and N-H str., Ar-NH), 3058 (C-H str., Ar-CH), 2944 (C-H str., ether), 1600 (C=O str. and N-H bending, 2˚ amide), 1510 (C=C skeletal str., phenyl), 1287 (C-O-C str., Ar-OCH₃).

 

N-(4-bromophenyl)-4-hydroxy-3-methoxy benzamide (C-13)

IR (KBr pellets) cm^-1:3438 (O-H str., Ar-OH), 3050 (C-H str., Ar-CH), 2940 (C-H str., ether), 1601 (C=O str. and N-H bending, 2˚ amide), 1508 (C=C skeletal str., phenyl), 670 (C-Br str., aromatic). ¹H NMR (CDCl₃, δppm): 6.79-7.55 (m ,7H, Ar-H), 8.01 (s, 1H, NH), 3.88 (s, 3H, OCH₃).

 

4-hydroxy-3-methoxy-N-(4-nitrophenyl) benzamide (C-14)

IR (KBr pellets) cm^-1: 3476 (O-H str., Ar-OH), 3360 (N-H str., Ar-NH), 2938 (C-H str., ether), 1598 (C=O str., 2˚ amide and N-H bending, 2˚ amide), 1503 ((NO₂ sym. str., Ar-NO₂).

 

In vitro evaluation of antioxidant activity:

In vitro evaluation of antioxidant activity of all the newly synthesized derivatives was done by DPPH free radical scavenging assay method (Woranuch et al., 2015⁴⁷). Concentration of the synthesized compounds that gave 50% inhibition was calculated from the graph plotted as compound concentration against percent inhibition. Compounds 8, 10 and 11 were found as most active antioxidants among all the synthesized compounds with IC₅₀ values of48.37 µg/ml, 48.84 µg/ml and 49.38 µg/ml respectively and may be considered as effective compounds for the development of new antioxidant agents.

 

Table 3: Antioxidant activity of  synthesised derivatives

 

In vitro evaluation of α-amylase inhibition:

The in vitro α-amylase inhibitory activity evaluation of all newly synthesized compounds (1-14) was performed using diastase and by colorimetric method (Nickavar et al. 2008³⁰) using acarbose as standard. Compounds 1, 5 and 8 were found as the most potent α-amylase inhibitor compounds among all the synthesized with IC₅₀ values of 55.83 µg/ml, 54.74µg/ml and 56.51µg/ml respectively. Results are presented in Table 4.

 

Table 4: α-Amylase inhibition activity of synthesized derivatives

^*Standard

 

Molecular docking study of α-amylase against newly synthesized derivatives:

All the synthesized compounds were subjected to molecular docking study using the AutoDock Tools (ADT) version 1.5.2 and Auto-Dock version 4.0. Three-dimensional structure of α-amylase was taken from protein data bank (pdb code 1HNY). From the docking study of all the compound 1, 5 and 8 were found to possess maximum binding with α-amylasehaving binding free energy of -40.03, -48.39 KJ/mol and -46.74 KJ/mol respectively which were comparable to  acarbose (standard) having binding free energy -43.43 KJ/mol.

 

 

Table 5: Relation between ΔG obtained after virtual screening to IC₅₀ value obtained after in vitro evaluation

S. No	Structure of ligand	IC50 value (µg/ml)	ΔGº (KJ/mol)
1.		55.83	-40.03
2.		57.66	-41.81
3.		63.04	-26.64
4.		60.11	-37.99
5.		54.74	-48.39
6.		61.93	-39.43
7.		66.56	-29.85
8.		56.51	-46.74
9.		64.68	-36.96
10.		67.93	-29.80
11.		61.91	-38.75
12.		63.08	-20.82
13.		67.49	-35.34
14.		59.44	-34.36
15*		54.61	-43.43

 

 

 

 

Fig 1: 3D and 2D diagrams of docked confirmation of compound 1 interacting with amino acid residues.

 

 

 

 

Fig2: 3D and 2D diagrams of docked confirmation of compound 5 interacting with amino acid residues.

 

Fig 3: 3D and 2D diagrams of docked confirmation of compound 8 interacting with amino acid residues.

Compound 1 formed one backbone hydrogen bond with ASP300 and one pi-pi stacking with TRP58 amino acid residues. Compound was found to form four hydrophobic interactions with LEU165, TYR62, TRP58 and TRP59 amino acid residues. Complex also formed three polar interactions with THR163, GLN63, ASN301 along with two negative charged interactions with ASP356 and ASP300 amino acid residues (Figure 1).

.

Compound 5 was found to form one backbone hydrogen bond with amino acid residue ASP300 along with one pi-pi stacking bond with TRP58. Complex also formed hydrophobic interactions with TYR62, LEU165, TRP59, TRP58 and TRP357 as well as two polar interactions with SER422 and SER421 amino acid residues. Compound also formed negative charged interactions with four amino acid residues namely GLU418, ASH730, GLU718 and GLU742 (Figure 2).

 

Compound 8 formed one backbone hydrogen bond with ASP300 as well as one pi-pi stacking bond with TRP58 amino acid residues. Compound also formed four hydrophobic interactions with amino acid residues namely LEU165, TRP59, TRP58 and TYR62 along with three polar interactions with GLN63, HIE101 and ASN301. Complex also formed two negative charged interactions with ASP356 and ASP300 (Figure 3).

 

In vitro evaluation of urease inhibitory activity:

All the newly synthesized compounds (1-14) were evaluated for their urease inhibition potential by indophenol method using thiourea as standard drug⁴⁸. Compounds 4 and 9 were found to show highest urease inhibition activity among all the newly synthesized compounds having IC₅₀ values of 69.01 µg/ml, 69.05 µg/ml respectively. Results are presented in Table 6. From results, it was observed that compounds 4 and 9 possess IC₅₀ value comparable to that of thiourea (positive control).

 

Table 6: Urease inhibition activity of newly synthesized derivatives.

*Standard

Molecular docking studies of urease towards newly synthesized derivatives:

Molecular docking is a computational study to generate and identify the most appropriate match between a macromolecule (ligand or small molecule) and its target. Docking process generally consists of three steps namely perception of the ligand and target, generation of putative complexes (sampling) and evaluating the fitness of the complex (scoring). All the synthesized compounds were subjected to molecular docking study using the AutoDock Tools (ADT) version 1.5.2 and Auto-Dock version 4.0. Crystallographic structure of Jack Bran Urease (pdb code 3LA4) having resolution 2.05 A⁰ was taken from protein data bank. 3D structure (PDB format) of synthesized derivatives were drawn by ChemSketch drawing package and docked with enzyme to observe the interaction of these compounds with target enzyme. Docking study revealed that compound 4 and 9 showed maximum binding towards urease with binding free energies of -48.95 KJ/mol and -59.77 KJ/mol respectively and were comparable with the standard drug (thiourea) taken having binding free energy -12.83 KJ/mol.

Table 7: Relation between ΔG obtained after virtual screening to IC₅₀ value obtained after in vitro evaluation

 

Fig 4: 3D and 2D diagrams of docked confirmation of compound 4 interacting with amino acid residues.

 

Fig 5:3D and 2D diagrams of docked confirmation of compound 9 interacting with amino acid residues.

 

Compound 4 formed a salt bridge with amino acid residue GLU 742 as well as three side chain hydrogen bond with amino acid residues LYS716, GLU718 and LYS709. Complex was found to form hydrophobic interaction with PHE712, VAL744, LEU13, PRO743, ALA16, ALA37, VAL36, LEU839 and TYR32 amino acid residues. Complex also formed three negative charge interaction with GLU718, ASH730 and GLU742 with a polar interaction with THR33 amino acid residue (Figure 4).

Compound 9 formed three side chain hydrogen bond with amino acid residues namely LYS716, ASH730 and TYR32 along with hydrophobic interactions with seven amino acid residues including PHE838, PHE712, VAL744, VAL36, TYR32, MET746 and PRO717. Compound formed four negative charged interactions with GLU418, ASH730, GLU718 and GLU742 amino acid residues. Compound had also showed two polar interactions with amino acid residues namely SER422 and SER421 (Figure 5).

 

Structure Activity Relationship studies (SAR):

From the antioxidant, α-amylase and urease inhibition screening results of 4-hydroxy-3-methoxy benzoic acid derivatives, the following structure activity relationship can be derived (Figure 6):

 

1. Substitution with chloro group (electron withdrawing group) at o-position at phenyl ring attached to hydroxybenzamide ring, as present in compound N-(2-chlorophenyl)-4-hydroxy-3methoxy benzamide (C-8), increases antioxidant activity.

 

2. Substitution with nitro group (electron withdrawing group) at o-position at phenyl ring attached to hydroxybenzamide ring, as present in compound 4-hydroxy-3-methoxy-N-(3-nitrophenyl) benzamide (C-5), increases α-amylase inhibitory activity.

 

3. Substitution with methoxy group (electron donating group) at m-position at phenyl ring attached to hydroxybenzamide ring, as present in compound 4-hydroxy-3-methoxy-N-(3-methoxyphenyl) benzamide (C-9), increases urease inhibitory activity.

 

From these results we may conclude that different structural requirements are important for different targets.

 

Fig 6: Structure activity relationship of synthesized derivatives.

 

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Received on 19.07.2019           Modified on 12.09.2019

Accepted on 21.11.2019         © RJPT All right reserved