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 International Journal of Medical Sciences and Pharma Research 

Open Access to Medical Science and Pharma Research

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Pharmacological Evaluation of Novel 1,3,4-Oxadiazole Derivatives

Aswathy Ramesh ¹*, Dr Rakesh Kumar Jat ², Dr R Arunkumar ³ 

¹ PhD scholar, JJT University, Rajasthan, Jhunjhunu, India

² HOD, Department of Pharmacy, JJT University, Rajasthan, Jhunjhunu, India

³ Principal, Holygrace Academy of Pharmacy, Thrissur, Kerala, India

Oxadiazoles (C2H2N2O) are heterocyclic compounds with a five membered ring as the basic nucleus in which three carbon atoms are replaced by heteroatoms. These compounds come under azole family in which 2 nitrogen atoms and one oxygen atom are present in the compound instead of carbon. By their multiple action in both medicinal as well as material chemistry, Oxadiazoles attracted the researcher’s attention⁵.

1, 3, 4-oxadiazoles are thermally stable neutral aromatic nuclei that are found to possess a broad spectrum of biological activities. They are associated with wide variety of pharmacological activities like antibacterial, antifungal, tuberculostatic, anticonvulsant, analgesic, anti-inflammatory, diuretic, antiemetic and insecticidal properties. Recently they were found to possess anti-inflammatory, antitumor and antiviral activities¹⁵. And hence, an intensified investigations are going on in the field of oxadiazoles due to tremendous outbreak in lifestyle diseases.

In the present study novel derivatives of 1,3,4-oxadiazole are synthesized and are subjected to pharmacological evaluation. Most of the derivatives showed promising activity. 

2.1 Chemicals

All the chemicals and reagents used in the present work were of analytical grade and obtained from Nice Chemicals, Mumbai and Chemco, Mumbai. 

2.2 Synthesis of compounds

30 analogues, that are selected from series designed using ChemDraw Ultra and Chem 3D Ultra, are subjected to molecular properties prediction using Molinspiration software. Those derivatives satisfying the Lipinski rule of five¹⁰ were then subjected to PASS software to predict the general biological potential of the novel proposed compounds. Derivatives with maximum PASS⁴ value for the desired activities are then synthesized in the laboratory using the selected scheme (given Fig 1) based on literature survey²⁰.

Step 1:

Step 2:

Step 3:

Figure 1:  Mechanism of synthesis

2.2.1 Synthesis of methyl salicylate¹³

A mixture of salicylic acid (0.47mol; 0.65g) and methanol (2ml) was taken in a 5ml round bottom flask. The flask was stirred well until the solid dissolves. Drop by drop addition of 0.75 ml of concentrated H2SO4 was done with constant agitation. The flask was then connected to a water condenser capped with a drying tube that has been loosely packed with CaCl2. Reflux the content for 75 min (80^oC). The solution was cooled to room temperature. Then extracted the solution with CHCl3 (1 ml x 3). The organic layer was collected. The combined solution was treated with 1 ml of aqueous 5% NaHCO3 solution. The solution was mixed well and was allowed to stand for separation the organic layer was collected and transferred to a dry vial. It was then allowed to evaporate over anhydrous Na2SO4. The product was purified.

2.2.2 Synthesis of salicyl hydrazide¹²

A mixture of methyl salicylate (0.1 mol; 15.2 ml) and hydrazine hydrate (0.2 mol; 10 ml) were boiled in 50 ml of 95% ethanol by connecting the flask to a condenser which promote flow back and reheating for 7-8 hrs. The water content in the succedent mixture was reduced, cooled and poured to crushed ice. The remaining mass thus separate out was filtered, dried and purified by recrystallization, mp.142-1440C.

2.2.3 Synthesis of Ox1-Ox4, Ox9, Ox10

A mixture of salicyl hydrazide (0.1mol; 1.52g) and carboxylic acid (0.1mol) was liquified in phosphorus oxy chloride (5ml) and boiled under reflux for 35 min. The liquid mixture was gently poured over broken ice and then neutralised with 5% NaHCO3. The solution was kept overnight. The separated solid mass was filtered, dried and purified by recrystallization using ethanol.

2.2.4 Synthesis of ethyl phenyl acetate²¹

A mixture of phenyl acetic acid (0.1 mol; 13.6 g), ethanol (0.4 mol; 18.4 ml) and concentrated H2SO4 (0.05 mol; 4.9 ml) were taken in a 50 ml round bottom flask and was refluxed for 3 hrs. The solution was cooled to room temperature, neutralised with 10% Na2CO3. The solution was extracted with diethyl ether (10 ml x 4) in a separating funnel. The upper ethereal layer was collected, purified by distillation on rotary evaporator.

2.2.5 Synthesis of phenyl acetic acid hydrazide

A mixture of ethyl phenyl acetate (15 ml) and methanol (10 ml) was taken in a 50ml round bottom flask. The reaction vessel was cooled to 0-5 0C. To this solution, hydrazine hydrate (15 ml) was added drop wise with occasional stirring. Then the solution was stirred for 60 min. After the evaporation of methanol, crude precipitate was collected and washed with n-hexane, mp: 113-1160C.

2.2.6 Synthesis of Ox5-Ox8

A mixture of phenyl acetic acid hydrazide (0.1 mol; 1.50 g) and different carboxylic acid (0.1 mol) was dissolved in phosphorus oxy chloride (5 ml). The mixture was refluxed for 35 min. The reaction mixture was slowly poured over crushed ice and then neutralized with 5% NaHCO3. The solution was kept overnight. The separated solid mass was filtered, dried and purified by recrystallization with ethanol.

The synthesized derivatives are 2-(5-(4-hydroxyphenyl)-1, 3, 4-oxaddiazol-2-yl) phenol (Ox1), 2-(5-styryl-1, 3, 4-oxadiazol-2-yl) phenol (Ox2), 2-(5-(4-chlorophenyl)-1, 3, 4-oxadiazol-2-yl) phenol (Ox3), 2-(5-(3, 5-dinitrophenyl)-1, 3, 4-oxadiazol-2-yl) phenol (Ox4), 2-benzyl-5-(3, 5-dinitrophenyl)-1, 3, 4-oxadiazol(Ox5), 2-benzyl-5-styryl-1, 3, 4-oxadiazol (Ox6), 1-(5-benzyl-1, 3, 4-oxadiazol-2-yl)-2-phenylethanamine (Ox7), 2-benzyl-5-(4-chlorophenyl)-1, 3, 4-oxadiazole (Ox8), [2-(5- ((2, 4- dichloro phenoxy)methyl)- 1, 3, 4- oxadiazol-2-yl) phenol (Ox9), [2- (5- (2-hydroxy-(phenyl))- [1, 3, 4]- oxadiazol- 2-yl) phenyl acetate(Ox10). Fig 2 shows the structures.

Ox1                Ox2 

Ox3           Ox4 

Ox5          Ox6     

Ox7        Ox8   

Ox9     Ox10 

Figure 2: Structures of new derivatives of 1,3,4-oxadiazoles

The structure of synthesized derivatives are confirmed by their spectral analysis. Finally, the derivatives were subjected to Docking studies using ArgusLab⁷ and Schrodinger software and those derivatives having high docking scores are evaluated for their desired biological activity in laboratory.

2.3 Pharmacological evaluation

2.3.1 Antibacterial Screening⁶

 The following micro-organisms were used to study the anti-bacterial activity:

Staphylococcus aureus - Gram positive organism.

Escherichia coli - Gram negative organism.

Nutrient agar broth was melted in an autoclave. The broth was then poured to petri-dishes (30 ml each) and allowed it to set. The culture media thus prepared was then inoculated with 0.5 ml of 24 hrs old culture. In each petri-dish, four wells of 6mm diameter were made at equal distance by digging the surface with an aseptic cork-borer and the separated agar was discarded. The sample solutions of 0.1ml volume having specific concentration were added to the wells using sterile micropipette. Separate petri-dishes were prepared for both standard and the control (DMF) and were noted. Amoxicillin (50 g/0.1 ml) were used as standard drug. The sample was allowed to spread in the agar medium by placing the petri dish in refrigerator for a time period of half an hour and then incubated at 37± 0.5⁰C for one day. The appearance of clear zone where microbial growth is inhibited is a sign of antibacterial activity. The dimension of clear zone was individually measured and the average value was calculated for each test compound. The results obtained are collated with that of reference drug amoxicillin. 

Tube dilution method¹⁴ is used to find minimum inhibitory concentration.

Procedure for Standardization of inoculum:100ml of inoculum was prepared by incorporating 1ml stock culture of bacterium in nutrient broth. It was then maintained at 37^oC for one day and allowed to incubate. Further growth was suppressed by incubating at 4^oC. 1ml of this culture was introduced to 9ml sterile water taken in another test tube. This dilution was carried to 10-fold serially from 10^-1 to 10^-10 dilution. 10^-7 to 10^-9 dilutions were taken and 0.2 ml of the diluted culture were inoculated on solid nutrient agar plate. The colony forming unit (CFU) was counted after incubation at 37^oC for 24 hrs. From this suitable inoculum load of cells/ml of selected broth was prepared. This selected broth is called working stock culture.

Procedure for Antibacterial activity: The new compounds were subjected to antibacterial studies using Staphylococcus aureus and E.coli. The standard solution of synthesized compounds was prepared in 1000 µg/ml concentration. 8 assay tubes were taken for each bacterium and 1ml working stock was added in each tube except in first tube. To this first tube, 1.8ml stock culture was added. It was then mixed thoroughly with 0.2ml of test solution and transfer 1 ml of this solution to the next tube. Repeating the step to get concentration of test solutions in the following order.100 microgram/ml, 50microgram/ml, 25microgram/ml, 12.5 microgram/ml, 6.25 microgram/ml, 3.125 microgram/ml and 1.5 microgram /ml. The standard drug Amoxicillin was prepared in the same manner to produce different dilutions ranging from 100µg -1.5µg/ml. The 1 ml working stock was taken as the positive control. 1 ml of solvent DMSO was taken as the negative control. The assay tubes were incubated at 37^oC for 24hrs and observation were done at end of 24hrs. With the aid of test sample containing lowest concentration that blocked the turbidity formation, inhibition can be determined after observing the amount of growth in the tube which cause cloudiness. Lowest concentration of antibiotic that caused complete inhibition of bacterial growth was taken as MIC of the compound against that microorganism. The cloudiness was examined after incubating the tubes for one day.

2.3.2 Anticancer activity¹⁶

In this method mice with tumour cells (Dalton’s lymphoma ascites cells) were selected and these cells from the peritoneal cavity was collected. Then using phosphate-buffered-saline or normal saline solution, these cells were thoroughly washed. The ability of these cells to survive was done by method using trypan-blue method. A suspension of alive cells with 1X105 cells in 0,1ml proportion was introduced to different test tubes each of which contain test compound with various concentration. By adding enough quantity of PBS, 1ml volume was attained. A tube was kept as a control in which no other additives except cell suspension was present. These mixtures were then allowed to incubate at a temperature of 37^oC for a time period of three hours. To this suspension 0.1ml trypan blue having 1% concentration was mixed and it was then placed on a haemocytometer immediately after standing for two to three minutes. When observed blue coloured cells give the number of dead cells as live cells fails to take up the colour. The result was calculated by counting separately the total number of coloured and colourless cells.

2.3.3 Antidiabetic activity²²

Test Procedure: The activity was determined using a modified assay of that described in the Worthington Enzyme Manual²³. 500ml of -amylase standard solution was prepared by dissolving 0.5mg/ml of the same in sodium phosphate buffer with molarity 0.02 and a pH of 6.9 was adjusted by the addition of 0.006 M NaCl and different concentrations (in μg) of extract as inhibitor were incubated initially around 10 minutes by maintaining a temperature of 37oC. Then each tube was added with another 500L of solution in which 1% starch dissolved in 0.02M sodium phosphate buffer having 6.9 pH value. It was again incubated for 5 more minutes at room temperature then 1.0 ml of indicator DNSA reagent was added to stop the reaction. After the addition, the tubes were placed in boiling water bar and allow to incubate. When the time reached 5 minutes, it was cooled until room temperature is obtained. Sufficient amount of distilled water was added to make the final volume to 10ml. with the aid of spectrophotometer operating in the UV-Visible range, the optical density was recorded at 540 nanometre wavelengths. The absorbance readings were compared with the controls and blank that contained buffer instead of sample extract.

Among the designed derivatives subjected to various software for their properties and activity prediction, ten derivatives are chemically synthesized in the laboratory. Of which, six derivatives (Ox1, Ox2, Ox3, Ox4, Ox9, Ox10) were synthesized by the reaction between salicylic acid and various aromatic acids and other four derivatives (Ox5, Ox6, Ox7, Ox8) were synthesized by using phenyl acetic acid as starting material. The synthesized derivatives were confirmed by IR, NMR and MASS spectral analysis¹⁷. Table 1, 2 and 3 shows the readings

Table 1: Characterization by IR

Table 2: Characterization by ¹HNMR

Table 3: Mass spectral data

3.1 Anti-bacterial effect of derivatives

Fig 3 and Fig 4,5 shows zone of inhibition and MIC respectively. 

Figure 3: Photo of Antibacterial activity of Ox4 on E. coli

Figure 4: Photos of MIC values of Amoxicillin

Figure 5: Photos of MIC values of Ox4

Table 4 and Table 5 shows the average restriction area in disc diffusion method and minimum inhibitory concentration in tube dilution method respectively.

Table 4: Antibacterial activities of synthesized compounds (disc diffusion method)

Readings are mean of three individual measurements

Table 5: Minimum Inhibitory Concentrations (MIC)

The derivatives Ox2 and Ox4 showed significant anti- bacterial activity against E.coli and Ox4 showed moderate inhibitory activity against Gram positive organism (S.aureus).

1. Anticancer activity of derivatives

Fig 6 shows graphical representation of cytotoxic activity of Ox8. Table 6 shows the reading of trypan blue exclusion method.

Figure 6: Cytotoxicity of Ox8 (trypan blue exclusion method)

Table 6: Cytotoxic effect of Ox8 (trypan blue exclusion method)

Compound Ox8 having high docking score (-11.3906 Kcal/mol) when docked with 3VO3, was evaluated for its in-vitro cytotoxicity against DLA cells and was found to have 52% cytotoxic activity at 200 µg/ml

Fig 7 and Fig 8 shows the antidiabetic activity of Ox10 and standard drug Acarbose respectively. Table 7 and 8 shows the corresponding readings.

Figure 7: Alpha amylase inhibitory activity of Ox10

Figure 8: Alpha amylase inhibitory activity of standard drug Acarbose

Table 7: Antidiabetic activity of Ox10 (by DNSA Method)

Table 8: Antidiabetic activity of standard drug Acarbose (by DNSA Method)

Among the 10 selected derivatives, Ox10 having high docking score (-8.8683 Kcal/mol) when docked with 1B2Y, was evaluated for its alpha-amylase inhibitory activity using DNSA method. Ox10 was found to have 81.53% of inhibition at 100g/ml when compared to standard Acarbose.

The detailed literature studies revealed that various oxadiazole derivatives have biological activities like anti-bacterial⁸, anti-fungal¹⁸, anti-inflammatory¹, anti-tubercular², anticancer⁹, anticonvulsant³, analgesic activities¹¹, antiviral activity¹⁹ etc.

In this study, a series of 2, 5-disubstituted- 1, 3, 4- oxadiazole derivatives were prepared from salicylic acid and phenyl acetic acid. The compounds Ox2 and Ox4 showed promising antibacterial activity against E.coli while Ox4 showed better activity compared to the standard drug amoxicillin at a concentration 500 µg/ml.

Result of docking studies showed that compound Ox8 (-11.3906 Kcal/mol) and Ox10 (-8.8683 Kcal/mol) with high docking scores possess good interaction with the enzyme and hence possess better anticancer and antidiabetic activity respectively when compared to other derivatives.

The compound Ox8 shows 52% cytotoxic activity against DLA cells at a concentration 200 g/ml when compared to standard drug 5-fluorouracil.

The compound Ox10 shows 81.53 % of inhibition at a dose of 100g/ml when compared to standard drug Acarbose which shows 61.63% of inhibition at a dose of 100µg/ml.

Acknowledgement 

Authors are grateful to Shri Jagdishprasad Jhabarmal Tibrewala University, and St. Josephs College of Pharmacy, Cherthala for providing the necessary facilities to carry out this research work.

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