SYNTHESIS, CHARACTERIZATION AND EVALUATION OF HEPATOCYTES REGENERATOR POTENTIALITY OF SOME NOVEL OXADIAZOLE DERIVATIVES FOLLOWED BY MOLECULAR DOCKING AGAINST NF-KB

SYNTHESIS, CHARACTERIZATION AND EVALUATION OF HEPATOCYTES REGENERATOR POTENTIALITY OF SOME NOVEL OXADIAZOLE DERIVATIVES FOLLOWED BY MOLECULAR DOCKING AGAINST NF-KB

Asish Bhaumik^*1 and M. Chinna Eswaraiah ²

Department of Pharmaceutical Sciences ¹, Assam down town University, Gandhi Nagar, Panikhaiti, Guwahati - 781026, Assam,  India.

Department of Pharmacognosy ², Anurag Pharmacy College, Ananthagiri, Kodad - 508206, Suryapet, Telangana, India.

ABSTRACT: NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex that controls transcription of DNA, cytokine production and cell survival. Incorrect regulation of NF-κB has been linked to cancer, inflammatory and autoimmune diseases and improper immune development. The main objective of the present research work was the synthesis of N-(4-{[5-(substituted phenyl)-1,3,4-oxadiazol-2-yl] methoxy}phenyl) acetamide derivatives and to evaluate the hepatocytes regenerator potentiality by molecular docking with 2V2T-NF-KB and  as well as In vivo methods and the synthesized compounds were characterized by IR, NMR, and Mass spectroscopy. The in-vivo Hepatoprotective activity was carried out by using albino rats where CCl₄ was used as a hepatotoxin. Molecular docking is performed to find out the binding affinity or molecular interaction energy (kcal/mol) of docked compounds. In silico molecular docking studies displayed the binding energies: -5.17, -5.52, -5.40, -4.60, -4.60, -4.87, -3.42, -3.85 k.cal/mol, of the synthesized compounds (AB1-AB8) which indicated that the compound had the high binding affinity towards the 2V2T-NF-KB protein and inhibit the NF-KB protein function in comparison with std. drug silymarin (-3.54 k.cal/mol). The in vivo experimental data displayed that the elevated levels of SGOT, SGPT, ALP and Sr. bilirubin were mainly due to CCl₄ intoxication, reduced significantly (*P<0.05) in rats, after treatment with synthesized compounds at dose of both 250 and 500 mg/kg body weight respectively. Although both the doses (250 and 500 mg/kg b. w.) of synthesized compounds executed in vivo hepatocytes regenerator potentiality, but the higher dose (500 mg/kg b. w.) was more effective and more significant.

NF-κB, transcription, IR, NMR, Hepatotoxin, Molecular docking etc

INTRODUCTION: In chemistry, methine is a trivalent functional group = CH− derived formally from methane. It consists of a carbon atom bound by two single bonds and one double bond, where one of the single bonds is to hydrogen.

The group is also called methyne or methene; its IUPAC systematic name is methylylidene or methanylylidene. Oxadiazole is derived from furan by replacement of two methine (-CH=) group by two pyridine type nitrogen (-N=). There are four possible isomers of oxadiazole forms ¹ (1, 2, 3, and 4) depending on the position of nitrogen atom in the ring and are numbered as shown in following figure.

Oxadiazole, a very weak base due to inductive effect of the extra heteroatom. The replace of two -CH= groups in furan by two pyridine type (-N=)

lowers aromaticity of resulting oxadiazole ring to an extent that the oxadiazole ring exhibit character of conjugated diene. The electrophillic substitutions in oxadiazole ring are extremely difficult at the carbon atom because, the relatively low electron density on the carbon atom which can be attributed to electron withdrawal effect of the pyridine type nitrogen atom. If oxadiazole ring is substituted with electron - releasing groups, the attack of electrophiles occurs at nitrogen. The ring is generally resistant to nucleophilic attack ¹.

 1                      2                   3                   4

1, 3, 4-oxadiazole is a five membered heterocyclic aromatic compound containing two nitrogen atom at position three and four and one oxygen atom present at position one. 1,3,4 oxadiazole is thermally stable than other oxadiazoles, these oxadiazole are very important compound in medicinal chemistry due to their biological activities, during last few years ².

1, 3, 4-oxadiazole

The various literature survey were reported that 1, 3, 4-oxadiazoles a class of five member heterocyclic compounds possessed a wide range of biological activities such as antimicrobial ³,  anti-inflammatory ⁴, antifungal ⁵, anticonvulsant ⁶, anthelmintics ⁷, analgesic ⁸, insecticidal ⁹, local anesthetic ¹⁰, antidiarrheal ¹, anticancer ¹²,  hypoglycaemic ¹³,  protease inhibitor-resistant HIV-1 ¹⁴, vasorelaxant ¹⁵ etc.

Experimental:

2.1. Chemistry:

2.1.1. MATERIALS AND METHODS: The chemicals which were used for the synthesis of target compounds and purification provided by Institutional store and were of AR and LR grade. The melting points of the synthesized compounds were determined by open capillary tube method. The IR spectra of the synthesized compounds were recorded on ABB Bomen FT-IR spectrometer MB 104 IR spectra recorded with potassium bromide pellets. The 1H-NMR spectra of synthesized compounds were recorded on instrument BRUKER NMR spectrometer in DMSO. The Mass spectra of

synthesized compounds were recorded JEOL GCmate. TLC method was used to determine the progress of the reaction. TLC plates are Pre-coated Silica gel (HF254-200 mesh) aluminium plates using ethyl acetate: n-hexane are used as solvent and visualized under UV- chamber. The IR, 1H-NMR and MASS spectra are used to assign the structure of synthesized compounds.

2.1.2. Steps involved in the synthesis of target compounds: ¹⁶

Step 1: Ethyl-4-acetamido phenoxy acetate: A mixture of p-acetamido phenol (0.01mol) and ethyl chloroacetate (0.01mol) was refluxed by using dry acetone in presence of anhydrous potassium carbonate (K₂CO₃) for 6hrs. The reaction mixture was cooled and then poured in to crushed ice. The solid product obtained, these product was filtered, dried and recrystallized using ethanol.

Step 2: 4-Acetamido phenoxy acetyl hydrazide: A mixture of ethyl-4-acetamido phenoxy acetate (0.01 mol), hydrazine hydrate (0.01 mol) in ethanol (15 ml) was refluxed for 5-8 hrs. The reaction mixture was cooled and then poured in to crushed ice. The solid product was obtained, this product was filtered, dried and recrystallized from ethanol.

Step 3: 2-(4-Acetamidophenoxy methyl) -5-aryl substituted - 1, 3, 4-oxadiazole ¹⁷: A mixture of 4-Acetamido phenoxy acetyl hydrazide (0.01mol) and various aromatic acids (0.01mol) in phosphorus oxychloride (10 ml) was refluxed for 6-8 hours. The completion of the reaction process was monitored by TLC plates. The contents were cooled and poured into the crushed ice and then neutralized the reaction mixture with sodium bicarbonate solution and the solid product was obtained, the product was filtered, dried and recrystallized from ethanol.

2.1.3. Synthetic Scheme:

Title compounds (AB1-AB8)

2.1.4. Spectral and other physical data of synthesized compounds:

Compound AB1: N-(4-{[5-(4-aminophenyl)-1,3,4-oxadiazol-2-yl]methoxy}phenyl) acetamide. M.F- C₁₇H₁₆N₄O₃, M.W 324.33, m. p 116⁰C, R_f – 0.77, Yield-74.5 %. IR (KBr) ν (cm^-1): 3393.16 cm^-1 (Ar-NH), 1633.67 cm^-1 (C=N), 1575.88 cm^-1 (C=C), 1069.05 cm^-1 (-C-O-C-), 3132.54 cm^-1 (Ar-CH), 1249.43 cm^-1(Ar-NH₂), ¹H-NMR δ (ppm): 6.45-7.4 (s, 8H, Ar-H), 5.17 (s, 2H,-CH₂), 4.1(s, 2H, -NH₂), 2.05 (s,1H, -CH₃), 8.05 (s, 1H, -NH), Mass (m/e value) % relative abundance: 324.12 (M⁺) (5.1), 310.87 (4), 296.22 (8.25), 282.76 (2.2), 272.38(2.32), 262.6432 (7.3), 248.34 (11), 217.12 (15), 207.14 (7), 116.67 (18), 58.33(B).

Compound AB2: N-(4-{[5-(2,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl]methoxy}phenyl) acetamide.. M.F- C₁₇H₁₃Cl₂N₃O₃, M.W 378.209, m. p 180 ⁰C, R_f –0.74, Yield-69.9 %. IR (KBr) ν (cm^-1): 3381.92 cm^-1 (Ar-NH), 1673.42 cm^-1 (C=N), 1545.03 cm^-1 (C=C), 1085.04 cm^-1 (-C-O-C-), 687.47 cm^-1 (C-Cl), 3115.62 cm^-1 (Ar-CH), ¹H-NMR δ (ppm): 6.6-7.82(s, 8H, Ar-CH), 2.5 (s, 3H, -CH₃), 8.03(s, 1H, -NH), 5.22(s, 2H, -CH₂), Mass (m/e value) % relative abundance: 377.03 (M⁺) (2.8), 333.16 (1.5), 325.42 (2.7), 286.43 (2.6), 183.26 (6), 160.62 (7), 140.65 (16), 115.64 (33),  95.53 (B).

Compound AB3: N-(4-{[5-(4-flurophenyl)-1,3,4-oxadiazol-2-yl]methoxy}phenyl) acetamide. M.F- C₁₇H₁₄FN₃O₃, M.W 327.309, m. p 189 ⁰C, R_f – 0.75, Yield-74 %. IR (KBr) ν (cm^-1): 3392.09 cm^-1 (Ar-NH), 1617.53 cm^-1 (C=N), 1528.16 cm^-1 (C=C), 1093.52 cm^-1 (-C-O-C-), 1371.78 cm^-1 (C-F),  3114.61 cm^-1 (Ar-CH), ¹H-NMR δ (ppm): 2.21 (s, 1H, -CH₃), 8.09 (s, 1H, -NH), 5.21(s, 1H, -CH₂), 6.7-8.01(m, 8H, Ar-CH), Mass (m/e value) % relative abundance: 327.10 (M⁺) (6.3), 310.37 (2.3), 299.57 (3), 282.87 (3.9), 266.22 (5), 249.61 (1.2), 232.72 (4), 104.86 (8.1),  75.50 (B).

Compound AB4: N-(4-{[5-(2-bromophenyl)-1,3,4-oxadiazol-2-yl]methoxy}phenyl) acetamide. M.F- C₁₇H₁₄BrN₃O₃, M.W 388.215, m. p 183 ⁰C, R_f – 0.65, Yield-69 %. IR (KBr) ν (cm^-1): 3286.82 cm^-1 (Ar-NH), 1617.53 cm^-1 (C=N), 1528.16 cm^-1 (C=C), 1093.52 cm^-1 (-C-O-C-), 687.47 cm^-1 (C-Br),  3114.61 cm^-1 (Ar-CH), ¹H-NMR δ (ppm): 2.21 (s, 1H, -CH₃), 8.09(s, 1H, -NH), 5.21(s, 1H, -CH₂), 6.7-8.01(m, 8H, Ar-CH), Mass (m/e value) % relative abundance: 387.02(M⁺) (6.3), 310.37 (2.3), 299.57 (3), 282.87 (3.9), 266.22 (5), 249.61 (1.2), 232.72 (4), 104.86 (8.1),  75.60 (B).

Compound AB5: N-(4-{[5-(2-bromo, 4 - nitro phenyl)-1,3,4-oxadiazol-2-yl] methoxy} phenyl) acetamide. M.F- C₁₇H₁₃BrN₄O₅, M.W 433.213, m. p 166 ⁰C, R_f – 0.64, Yield-60 %. IR (KBr) ν (cm^-1): 3381.95 cm^-1 (Ar-NH), 1684.44 cm^-1 (C=N), 1586.2 cm^-1 (C=C), 1064.25 cm^-1 (-C-O-C-), 1365.57 cm^-1(N=O), 619.89 cm^-1 (C-Br), 3130.43 cm^-1 (Ar-CH), ¹H-NMR δ (ppm): 6.74-8.36(m, 7H, Ar-CH) ,5.31(s, 2H, -CH₂),2.31 (s, 1H, -CH₃), 8.16(s, 1H, -NH), Mass (m/e value) % relative abundance: 432.00 (M⁺) (4), 388.71 (8.1), 362.27 (4.2), 233.28 (5), 217.31 (8.9), 182.52 (5), 96.79 (7), 78.82(B).

Compound AB6: N-(4-{[5-(4-nitrophenyl)-1,3,4-oxadiazol-2-yl]methoxy}phenyl)acetamide. M.F- C₁₇H₁₄N₄O₅, M.W 354.31, m. p 171 ⁰C, R_f – 0.72, Yield-64%. IR (KBr) ν (cm^-1): 3382.43 cm^-1 (Ar-NH), 1703.01 cm^-1 (C=N), 1592.32 cm^-1 (C=C), 1088.54 cm^-1 (-C-O-C-), 1378.11 cm^-1 (N=O), 3112.69 cm^-1 (Ar-CH), ¹H-NMR δ (ppm): 6.41-7.8(m, 8H, Ar-CH), 2.42 (s, 3H, -CH₃), 8.13(s, 1H, -NH), 5.21(s, 2H, CH₂), Mass (m/e value) % relative abundance: 354.09 (M⁺) (3.8), 335.16 (4.8), 302.39 (3.1), 287.43 (3.7), 249.58 (7.1), 226.00 (5.8), 204.96 (6.7), 127.56 (13.1), 103.69 (9),   89.93 (B).

Compound AB7: N-(4-{[5-(3,5-dinitrophenyl)-1,3,4-oxadiazol-2-yl]methoxy}phenyl) acetamide. M.F- C₁₇H₁₃N₅O₇, M.W 399.31, m. p 204 ⁰C, R_f – 0.68, Yield-78%. IR (KBr) ν (cm^-1): 3382.02 cm^-1 (Ar-NH), 1677.79 cm^-1 (C=N), 1530.6 cm^-1 (C=C), 1089.68 cm^-1 (-C-O-C-), 1372.45 cm^-1 (N=O),  1523.12 asym cm^-1 (N=O), 3117.5 cm^-1 (Ar-CH), ¹H-NMR δ (ppm): 6.83-8.42(m, 8H, Ar-CH), 5.35(s, 2H,-CH₂), 2.07 (s, 1H,- CH₃), 8.24 (s, 1H, -NH), Mass (m/e value) % relative abundance: 399.08 (M⁺) (5), 388.76 (13), 380.25 (8), 261.63 (8), 182.52 (5), 167.62 (17), 156.56 (19), 81.97(B).

Compound AB8: N-(4-{[5-(2-hydroxy-3,5-dinitro phenyl) - 1,3,4 - oxadiazol-2-yl] methoxy} phenyl) acetamide. M.F- C₁₇H₁₃N₅O₈, M.W 415.31, m. p 215 ⁰C, R_f – 0.72, Yield-68%. IR (KBr) ν (cm^-1): 3118.84 cm^-1 (Ar-NH), 1654.42 cm^-1 (C=N), 1541.89 cm^-1 (C=C), 1368.45 cm-1 (N=O), 1528.45 asym. cm-1 (N=O),1090.01 cm^-1 (-C-O-C), 3118.84 cm^-1 (Ar-CH), 3382.83 cm^-1(Ar-OH), ¹H-NMR δ (ppm): 6.7-7.6(s, 6H, Ar-CH), 2.11 (s, H, -CH₃), 8.00(s, 1H, -NH), 5.12(s, 1H, -CH₂), Mass (m/e value) % relative abundance: 415.07(M) (11.1), 318.68 (16), 292.76 (7), 276.89 (20), 249.99 (8.2), 236.0277 (28.1), 203.2266 (76), 182.2587 (8), 134.4966 (32), 116.55 (B).

2.2. Pharmacology:

2.2.1. Experimental animals and standard drug: White male albino Wister rats weighing about 200-250gm was used. They were obtained from the animal house of Anurag Pharmacy College, Kodad-508206, Telangana state.They were kept under observation for about 7 days before onset of experiment to exclude any intercurrent infection, had free access to normal diet and water. The experimental protocol was approved by IAEC (Institutional Animal Ethics Committee) of CPCSEA: 1712/P0/a/13/CPCSEA. The standard drug silymarin was purchased from retail local shop.

2.2.2. Molecular docking ¹⁸: Molecular docking is defined as an optimization problem, which would describe the “best-fit” orientation of a ligand that binds to a particular protein of interest. During the course of the process, the ligand and the protein adjust their conformation to achieve an overall “best-fit” and this kind of conformational adjustment resulting in the overall binding is referred to as “induced fit. The aim of the molecular docking to achieve an optimized conformation for both the protein and the ligand and to achieve relative orientation between protein and ligand such that free energy of overall system is minimized. The application of docking are the hit identification – docking combined with a scoring function can be used to quickly screen large databases of potential drugs in silico to identify molecules that are likely to bind to protein target of interest and the lead optimization – docking can be used to predict in where and in which relative orientation a ligand binds to a protein. This information may in turn be used to design more potent and selective analogues.

2.2.3. Computational Analysis: For the present research work (Project Id: RSID001020/10/2015, PDB Code: 2V2T) the Crystalline structure of the target protein NF-KB with PDB id 2V2T was retrieved from protein data bank and protein clean-up process was done and essential missing hydrogen atom were been added. Different orientation of the lead molecules AB1 to AB8 along with standard drug silymarin with respect to the target protein was evaluated by Autodock program and the best dock pose was selected based on the interaction study analysis.

FIG. 1: STRUCTURE OF PROTEIN NF-KB

2.2.4. Evaluation of acute toxicity: In the present study acute oral toxicity of the synthesized compounds were performed by acute toxic class method according to OECD guideline - 423 ¹⁹. In this method the toxicity of synthesized compounds were tested using a step wise procedure, each step using three rat of single sex (female/male). The rats were fasted prior to dosing (food but water should be with held) for three to four hours. Following the period of fasting the animal should be weighted and synthesized compound were administered orally at a dose 2000 mg/kg body weight.

Animals were observed individually after dosing at least once during the first 30min; periodically during the first 24 h with special attention giving during the first 4 h and daily thereafter, for total of 14 days. As know mortality observed with the above dose. Test compound dose reduced by specific intervals. The mortality was not observed at the dose 2000 mg / kg. So 250 mg /Kg body weight (low dose) and 500 (high dose) were selected for their pharmacological evaluation.

2.2.5. Experimental protocol for Hepatoprotetive activity: ²⁰ A total of 66 rats were taken and divided into 11 groups of 6 rats each.

Treatment Groups:

(A) Group I:   Normal Control Group (only the vehicle (1 ml/kg/day of 1% CMC; p.o.)

(B) Group II:   Negative Control CCl₄ 1 ml/kg (1:1 of CCl₄ in olive oil) i.p.

(C) Group III-X (AB1-AB8): (i) Low Dose Group [CCl₄ 1 mL/kg (1:1 of CCl₄ in olive oil)  i. p  + Synthesized compounds (250 mg/ kg b. w., p. o)]. Treatment was given daily for seven days orally and (ii) High Dose Group [CCl₄ 1 mL/kg (1:1 of CCl₄ in olive oil) i.p + Synthesized compounds (500 mg/ kg b. w., p. o)]

(D)  Group XI: Positive Control/Standard Group [CCl₄ 1 ml/kg (1:1 of CCl₄ in olive oil) i.p.+ Standard Silymarin 100 mg/kg orally ( p. o) for 7 days].

Collection of blood: On the 8th day, blood was collected by retro orbital puncture, under mild ether anesthesia after 8 hr fasting. Blood samples were centrifuged at 3000 rpm for 20 mins. Serum was separated and stored at – 20 °C until biochemical estimation was carried out.

Biochemical Analysis:

The Serum samples were analyzed for

(I)  Alanine Aminotransferase (ALT) (SGPT)

(II)  Aspartate Aminotransferase (AST) (SGOT)

(III) Alkaline Phosphatase (ALP)

(IV) Serum Bilirubin

Histopathological Analysis: The pathological status of hepatocytes of rat was done by FNAB ²¹.

RESULTS AND DISCUSSION:

3.1. Chemistry: The synthesis of target compounds (AB1-AB8) N-(4-{[substituted phenyl)-1,3,4-Oxadiazole-2-yl]methoxy}phenyl) acetamide were carried out by reacting Para acetamidophenol, ethylchloro acetate, hydrazine monohydrate and various aromatic acids. The synthesized compounds were characterized by IR, NMR, and Mass spectroscopy. The progress of the reaction was monitored by TLC using solvent systems of different polarities. TLC plates are pre-coated silica gel (HF254-200 mesh) aluminium and spots were visualized under U.V chamber and   the proposed structures of the synthesized compounds were ascertained by spectral data.

All the synthesized compounds having the following solubility profile: Insoluble in water, slightly soluble in chloroform, ethanol, and freely soluble in DMF, DMSO.

3.2. Molecular docking studies: Most of the scoring functions in molecular docking are physics-based molecular mechanics force fields that estimate the energy of the binding pose; a low (negative) energy indicates a stable system and thus a likely binding interaction. Molecular docking is performed to find out the binding affinity or molecular interaction energy (kcal/mol) of docked compounds. Lowest (negative value) energy of docked molecule indicates high binding affinity with the target protein/compound.

In silico molecular docking studies displayed the binding energies (Table 1 and Fig. 1- 6) : -5.17, -5.52, -5.40, -4.60, -4.60, -4.87, -3.42, -3.85 k.cal/mol,  of the synthesized compounds (AB1-AB8) which indicated that the compound had high binding affinity towards the 2V2T-NF-KB protein and inhibit the NF-KB protein function in comparison with standard drug silymarin (-3.54 k.cal/mol).

TABLE 1: FOR THE RESULTS OF MOLECULAR DOCKING OF SYNTHESIZED COMPOUNDS (AB1-AB8) & SILYMARIN WAS USED AS A STANDARD DRUG

Silymarin with 2V2T-NF-KB            Interaction Study                         Receptor Ligand Complex

FIG. 2: SILYMARIN WITH 2V2T-NF-KB INTERACTION

AB1 WITH 2V2T-NF-KB                                   AB2 WITH 2V2T-NF-KB

INTERACTION STUDY                                 INTERACTION STUDY

RECEPTOR LIGAND COMPLEX                 RECEPTOR LIGAND COMPLEX

FIG. 3: COMPOUND AB1 AND AB2 WITH 2V2T-NF-KB INTERACTION

 AB3 WITH 2V2T-NF-KB                             AB4 WITH 2V2T-NF-KB

INTERACTION STUDY                                  INTERACTION STUDY                                                                                

RECEPTOR LIGAND COMPLEX                RECEPTOR LIGAND COMPLEX

FIG. 4: COMPOUND AB3 AND AB4 WITH 2V2T-NF-KB INTERACTION

AB5 WITH 2V2T-NF-KB                            AB6 WITH 2V2T-NF-KB

INTERACTION STUDY                               INTERACTION STUDY

RECEPTOR LIGAND COMPLEX             RECEPTOR LIGAND COMPLEX

FIG. 5: COMPOUND AB5 AND AB6 WITH 2V2T-NF-KB INTERACTION

AB7 WITH 2V2T-NF-KB                                  AB8 WITH 2V2T-NF-KB

INTERACTION STUDY                                    INTERACTION STUDY

RECEPTOR LIGAND COMPLEX          RECEPTOR LIGAND COMPLEX

FIG. 6: COMPOUND AB7 AND AB8 WITH 2V2T-NF-KB INTERACTION

3.3. Evaluation of hepatoprotective activity:

Statistical analysis: The data (Table 2) were expressed as mean ± SD. Statistical differences at *P < 0.05 between the groups were analyzed by one-way ANOVA followed by Dunnett's Multiple Comparison Test using Graph Pad Prism 5.04 Instate software package. The data’s were compared with group 2 i.e. Negative Control group.

TABLE 2: FOR THE ASSESSMENT OF BIOCHEMICAL PARAMETERS

Biochemical analysis: The present study displayed that synthesized compounds were possessed a significant hepatoprotective activity. The declining of plasma enzyme level (Fig. 7-16) is a prognostic status of the hepatoprotective action of the drug. Protection of hepatic damage caused by carbon tetrachloride administration was observed by recording SGOT, SGPT, SALP and Serum bilirubin levels in different groups. The transport function of the hepatocytes is disturbed in hepatic injury, causing the leakage of enzymes due to altered membrane permeability. Although both the doses (250 and 500 mg/kg b. w.) of synthesized compounds executed hepatoprotective activity, the higher dose (500 mg/kg b. w.) is more effective and more significant. The in vivo experimental data displayed that the elevated levels of SGOT, SGPT, ALP and Sr. bilirubin were mainly due to CCl₄ intoxication,  reduced significantly (*P<0.05) in rats, after treatment with synthesized compounds.

FIG. 7: SGOT LEVEL (IU/L) IN DIFFERENT GROUPS AFTER TREATED WITH LOW DOSE OF TEST COMPOUNDS

FIG. 8: SGOT LEVEL (IU/L) IN DIFFERENT GROUPS AFTER TREATED WITH HIGH DOSE OF TEST COMPOUNDS

FIG. 9: SGPT LEVEL (IU/L) IN DIFFERENT GROUPS AFTER TREATED WITH LOW DOSE OF TEST COMPOUNDS

FIG. 10: SGPT LEVEL (IU/L) IN DIFFERENT GROUPS AFTER TREATED WITH HIGH DOSE OF TEST COMPOUNDS

FIG. 11: SALP LEVEL (IU/L) IN DIFFERENT GROUPS AFTER TREATED WITH LOW DOSE OF TEST COMPOUNDS

FIG. 12: SALP LEVEL (IU/L) IN DIFFERENT GROUPS AFTER TREATED WITH HIGH DOSE OF TEST COMPOUNDS

FIG. 13: SR. BILIRUBIN (mg/dl) IN DIFFERENT GROUPS AFTER TREATED WITH LOW DOSE OF TEST COMPOUNDS

FIG. 14: SR. BILIRUBIN (mg/dl) IN DIFFERENT GROUPS AFTER TREATED WITH HIGH DOSE OF TEST COMPOUNDS

FIG. 15: GRAPHICAL REPRESENTATION OF PERCENTAGE (%) OF DECLINED LEVEL OF ANALYTES IN DIFFERENT GROUPS AFTER TREATED WITH LOW DOSE OF TEST COMPOUNDS

FIG. 16: GRAPHICAL REPRESENTATION OF PERCENTAGE (%) OF DECLINED LEVEL OF ANALYTES IN DIFFERENT GROUPS AFTER TREATED WITH HIGH DOSE OF TEST COMPOUNDS

Treatment with a synthesized compounds (AB1-AB8) at a dose of 250 mg/kg b. w. decreased the SGOT: 10.76%, 8.74%, 9.08%, 7.16%, 9.58%, 6.61%, 11.65%, 7.80%, SGPT: 23.30%, 23.35%, 22.87%, 23.78%, 23.20%, 22.87%, 23.01%, 23.92%, ALP: 10.18%, 9.92%, 10.30%, 10.20%, 9.33%, 10.56%, 8.80%, 9.56% and Serum bilirubin levels by 36.98%, 42.46%, 46.57%, 36.98%, 38.35%, 42.46%, 36.98%, 38.35%, (significantly) respectively, while at higher dose of 500 mg/kg b. wt. was more effective, causing a reduction of SGOT: 25.33%, 24.69%, 24.83%, 23.85%, 24.69%, 23.75%, 26.22%, 24.19%, SGPT: 42.26%, 41.69%, 41.97%, 42.39%, 41.54%, 41.49%, 41.40%, 42.40%, SALP: 22.66%, 22.58%, 22.58%, 22.35%, 22.35%, 22.56%, 22.30%, 22.33%, and Sr. bilirubin: 54.79%, 55.10%, 57.46%, 57.68%, 53.51%, 55.83%, 55.04%, 53.85%. Silymarin is used as standard drug showed a significant reduction of level of SGOT: 54.79%, SGPT: 47.61%, SALP: 60.39% and Sr. bilirubin: 78.08% respectively receiving CCl₄ alone.

Histopathological analysis: The results of FANB of liver cells of rats of control, CCl₄ treated and treated with synthesized compounds (AB1-AB8) were represented in (Fig. 17-26). FANB of liver cells of rats revealed that the liver treated with CCl₄ showed a high degree of damage characterized by piecemeal necrosis and portal tract necrosis, interface hepatitis due to expanded portal tract by infiltration of lymphocytes, plasma cells and macrophages, fulminant necrosis which is characterized by submassive and massive necrosis.

FIG. 17: HEMATOXYLIN AND EOSIN STAINING, X 400:  (A) Normal control rat liver section (Group-I): Normal histological findings in the liver parenchyma; (B) Liver section of the rat intoxicated with Carbon tetrachloride (Group-II): Piecemeal and portal tract necrosis and mixed inflammatory infiltration; (C) Liver section of the rat treated with silymarin and intoxicated with carbon tetrachloride, showing mild necrotic debris. (Group-XI) (D) Liver section of the rat treated with 500 mg/kg of synthesized compound AB1 and intoxicated with carbon tetrachloride (Group-III): Showing healing of hepatic damage without necrosis.  C.V: Central vein, V.C: Vacuole, S.S: Sinusoidal spaces, H.C: Hepatocytes

FIG. 18: HEMATOXYLIN AND EOSIN STAINING, X40: (A) Normal control (Group-I): Showing normal histological findings in the liver parenchyma. (B) Liver section of the rat intoxicated with Carbon tetrachloride (Group-II): Showing submassive confluent necrosis, portal tract necrosis and mixed inflammatory infiltration. (C) Liver section of the rat treated with silymarin and intoxicated with carbon tetrachloride (Group-XI): Indicated reduced perivenular microvesicular steatosis without necrosis. (D) Liver section of the rat treated with 500 mg/kg of synthesized compound AB2 and intoxicated with carbon tetrachloride (Group-IV): Showing regeneration of hepatocytes and disappearance of necrotic debris.

FIG. 19: STAINED WITH HEMATOXYLIN AND EOSIN(original magnification×100, calibration bar = 100μm). (A) Normal control (Group-I): Showing normal histological findings in the liver parenchyma. (B) Liver section of the rat intoxicated with Carbon tetrachloride (Group-II): Showing intralobular Nectrotic debris, degeneration of portal vessel and inflammatory infiltration. (C) Liver section of the rat treated with silymarin and intoxicated with carbon tetrachloride (Group-XI): Indicated regeneration in sub massive necrosis is more orderly and may result in restoration of normal architecture. (D) Liver section of the rat treated with 500 mg/kg of synthesized compound AB3 and intoxicated with carbon tetrachloride (Group-V): Showing restoration of normal hepatic architecture

FIG. 20: HEMATOXYLIN AND EOSIN STAINING, X 400:  (A) Normal control rat liver section (Group-I): Normal histological findings in the liver parenchyma; (B) Liver section of the rat intoxicated with Carbon tetrachloride (Group-II): Indicated variable degree of bridging necrosis of hepatocytes, most marked in centrilobular and mononuclear cellular infiltrate in the lobule. Mild degree of liver cell necrosis is seen as ballooning degeneration (C) Liver section of the rat treated with silymarin and intoxicated with carbon tetrachloride (Group-XI) (D) Liver section of the rat treated with 500 mg/kg of synthesized compound AB4 and intoxicated with carbon tetrachloride (Group-VI): Showing centrilobular regeneration with restoration of c.v, ss and hepatocytes with mild necrosis. C.V: Central vein, V.C: Vacuole, S.S: Sinusoidal spaces, H.C: Hepatocytes.

FIG. 21: STAINED WITH HEMATOXYLIN AND EOSIN: (E) Liver section of the rat treated with 250mg/kg of synthesized compound AB1, AB2, AB3 and AB4 (Group: III, IV, V and VI) and intoxicated with carbon tetrachloride.

FIG. 22: HEMATOXYLIN AND EOSIN STAINING, X400:  (A) Normal control rat liver section (Group-I): Normal histological findings in the liver parenchyma; (B) Liver section of the rat intoxicated with Carbon tetrachloride (Group-II): Indicated focal of necrosis of hepatocytes, most marked in centrilobular and mononuclear cellular infiltrate in the lobule with varying portal tract inflammation.. Mild degree of liver cell necrosis is seen as ballooning degeneration; (C) Liver section of the rat treated with silymarin and intoxicated with carbon tetrachloride (Group-XI); (D) Liver section of the rat treated with 500 mg/kg of synthesized compound AB5 and intoxicated with carbon tetrachloride (Group-VII): Showing centrilobular regeneration with restoration of c.v, ss and hepatocytes with mild to moderate necrotizing cells.  C.V: Central vein, V.C: Vacuole, S.S: Sinusoidal spaces, H.C: Hepatocytes.

FIG. 23: HISTOPATHOLOGY REPORT. (A) NORMAL CONTROL RAT LIVER SECTION (Group-I): Showed a normal lobular architecture of the liver; (B) Liver section of the rat intoxicated with Carbon tetrachloride (Group-II): Showed moderate panacinar necrosis and inflammation and neutrophil infiltration also observed in the centrilobular region with portal triaditis; (C) Liver section of the rat treated with silymarin and intoxicated with carbon tetrachloride (Group-XI): Showed minimal inflammation and hepatic congestion with moderate portal triditis and their lobular architecture was normal; (D) Liver section of the rat treated with 500 mg/kg of synthesized compound AB6 and intoxicated with carbon tetrachloride (Group-VIII): Showed minimal inflammation with moderate portal triditis, mild necrosis and their lobular architecture was normal.

FIG. 24: HISTOPATHOLOGY REPORT. (A) NORMAL CONTROL RAT LIVER SECTION (Group-I): Showed a normal structure of the liver; (B) Liver section of the rat intoxicated with Carbon tetrachloride (Group-II): Showed portal inflammation and panacinar necrosis necrosis; (C) Liver section of the rat treated with silymarin and intoxicated with carbon tetrachloride (Group-XI): Showed minimal portal inflammation and their lobular architecture was normal; (D) Liver section of the rat treated with 500 mg/kg of synthesized compound AB7 and intoxicated with carbon tetrachloride (Group-IX): Showed minimal inflammation with moderate portal triditis, moderate necrotic debris and their lobular architecture was normal.

FIG. 25: AT A MAGNIFICATION OF 200 X OF HEMATOXYLIN AND EOSIN-STAINED LIVER SECTIONS. (A) Normal control rat liver section (Group-I): No noticeable histological changes; (B) Liver section of the rat intoxicated with Carbon tetrachloride (Group-II): Showed portal inflammation and confluent necrosis; (C) Liver section of the rat treated with silymarin and intoxicated with carbon tetrachloride (Group-XI): Showed minimal portal inflammation and their lobular architecture was normal; (D) Liver section of the rat treated with 500 mg/kg of synthesized compound AB8 and intoxicated with carbon tetrachloride (Group-X): Showed minimal inflammation with moderate necrosis and portal triditis and their lobular architecture was normal.

FIG. 26: STAINED WITH HEMATOXYLIN AND EOSIN: (E) Liver section of the rat treated with 250mg/kg of synthesized compound AB5, AB6, AB7 and AB8 (Group: VII, VIII, IX and X) and intoxicated with carbon tetrachloride.

TABLE 3: FOR HEPATITIS ACTIVITY SCORE: NECRO INFLAMMATORY ACTIVITY

Histopathological scoring: ²² As prognostic indicator of chronic hepatitis, criteria have been evolved to classify chronic hepatitis by giving “hepatitis activity score” ranging from none to minimal/mild to moderate and severe describe by  Knodell and Ishak based on the following features (Table 3, 4 )

Necroinflammatory activity:

(i) Periportal necrosis i.e.: piece necrosis and / or bridging necrosis: Ranging from score 0 as “no necrosis” to score 4 as “multilobular necrosis”.

 (ii) Intralobular necrosis, focal or confluent: Ranging from score 0 as “none” to score 4 for “<10 focai” for focal necrosis and score 6 as “panacinar/ multiacinar” for confluent necrosis. (iii) Extent and depth portal inflammation: Ranging from grade 0 as “no inflammation” to grade 4 having “marked portal inflammation”.

Stage of fibrosis: Extent and density of fibrosis: Ranging from score 0 as “no fibrosis to score 6 as “cirrhosis”.

TABLE 4: FOR HEPATITIS ACTIVITY SCORE: PORTAL INFLAMMATORY ACTIVITY

CONCLUSION: The above experimental data concluded that the synthesized compounds had the potential hepatocytes regenerator ability which was proved by Hepatitis Activity Score (HAC) shown in vivo and in silico molecular docking studies of synthesized compounds were revealed comparable binding energies and similar docking poses on target proteins such as 2V2T-NF-KB and known to be inhibitors of NF-KB.

ACKNOWLEDGEMENTS: We would like to thank the Department of Pharmacology and Pharmaceutical Biotechnology, Anurag Pharmacy College, Kodad, Telangana. We would like to thank Noble Research Solutions, Chennai, a research centre for computational chemistry and molecular docking for carrying out the docking studies.

CONFLICT OF INTEREST: We declare that we have no conflict of interest.

REFERENCES:

1. Clapp LB, Katritzky AR, Ress CW. (Eds.), “1, 2, 3-oxadiazoles and 1, 2, 4-oxadiazoles”. Comprehensive Heterocyclic Chemistry, Pergamon Press, Oxford, 1984; 6:365.
2. Hetzheim A., Mockel K., “1, 3, 4-oxadiazole”. Adv. Heterocycl. Chem. 1966; 7:183.
3. Neeraj Kumar Fuloria, Vijender Singh, Mohammad Shaharyar and Mohammad Ali. “Synthesis and Antimicrobial Evaluation of Some New Oxadiazoles Derived from Phenylpropionohydrazides”. 2009; 14:1897-1903.
4. Shalabh Sharma, Virendra Kishor Srivastava & Ashok kumar, “Synthesis of some newer indolyl-thiadiazolyl-pyrazolines and indolyl-oxadiazolyl-pyrazolines as potential anti-inflammatory agents”. J. Chem., 2002; 41B: 2647-2654.
5. Cai-Jun Chen, Bao-An Song, Song Yang, Guang-Fang Xu, Pinaki S. Lin-Hong Jin, De-Yu Hu, Qian-Zhu Li, Fang Liu, Wei Xue, Ping Lu and Bhadury, Zhuo Chen., “Synthesis and antifungal activities of 5-(3,4,5-trimethoxyphenyl)-2-sulfonyl-1,3,4-thiadiazole and 5- (3, 4,5-trimethoxyphenyl)-2-sulfonyl - 1, 3, 4-oxadiazole derivatives”. Bioorg. Chem. 2007; 15: 3981–3989.
6. Almasirad A, Tabatabai S.A, Faizi M, Kebriaeizadeh A, Mehrabai N, Dalvandi A and Shafiee A. “Synthesis and anticonvulsant activity of new 2-substituted-5-[2-(2-fluorophenoxy)phenyl]-1,3,4-oxadiazoles and 1,2,4-triazoles”. Med. Chem. Lett. 2004; 14:6057–6059.
7. Husain M.I and Shukla M.K. “Search for potent anthelmintics. Part VIII: 4-substituted-7-coumarinyl oxyacetyl thiosemi carbazides and 7-coumarinyloxy methyl -1,2,4-triazoles and 1,3,4-oxadiazoles”. Indian Chem. Soc. 1978; 55:826–828.
8. Vasudeva Adhikari. ”Synthesis of some new 2-(3-methyl-7-substituted -2-oxoquinoxyalinyl)-5-(aryl1)-1, 3, 4-oxadiazoles as potential non-steroidal anti-inflammatory and analgesic agents”. J. Chem. 2008; 47B: 439-448.
9. Cao S, Qian X, Song G and Huang Q. “Synthesis and insecticidal activity of new 2-(5-(trifluoromethyl) pyri dyloxymethyl)-1,3,4-oxadiazoles”. Fluorine Chem. 2002; 117:63–66.
10. Rajak H, Kharya MD and Mishra P. “Synthesis and Local Anesthetic Activity of Some Novel N-[5-(4-Substituted) phenyl-1,3,4-oxadiazol-2-yl]-2-(Substituted)-Acetamides”. Arch. Pharm. 2008; 341(4):247–261.
11. Adelstein GW, Yen CH, Dajani EZ and Bianchi RG. “3, 3-diphenyl-3-(2-alkyl-1,3,4-oxadiazol-5-yl) - propylcyclo akyl amines, a novel series of antidiarrheal agents”. Med. Chem. 1976; 19(10):1221–1225.
12. Aboraia A.S, Abdel-Rahman H.M, Mahfouz N.M and E.L-Gendy M.A. “Novel 5-(2-hydroxyphenyl)-3-substituted-2, 3-dihydro-1, 3, 4-oxadiazole-2-thione derivatives: Promising anticancer agents”. Med. Chem. 2006; 14:1236–1246.
13. Hanna MA, Girges MM, Rasala D and Gawinecki R. “Synthesis and pharmacological evaluation of some novel 5-(pyrazole-3-yl)thiadiazole and oxadiazole derivatives as potential hypoglycemic agents”. Arzneim-Forsh. 1995; 45:1074–1078.
14. Kim RM, Rouse EA, Chapman KT, Schleif WA, Olsen DB, Stahlhut M, Rutkowski CA, Emini EA and Tata JR. P1'.” Oxadiazole protease inhibitors with excellent activity against native and protease inhibitor-resistant HIV-1”. Med. Chem. 2004; 14:4651–4654.
15. Girish R. Bankar, K. Nandakumar, Pawan G. Nayak, Anjali Thakur, Mallikarjuna Rao Chamallamudi, Gopalan Kutty Nampurath., “Vasorelaxant effect in rat aortic rings through calcium channel blockage: A preliminary in vitro assessment of a 1,3,4-oxadiazole derivative”. Chemico-Biological Interactions 2009; 181: 377–382.
16. K . B:868-872.nt synthesis of 1,3,4-oxadiazolyl 1,8-naphthridines under microwave irradiation" Ilango, P Valentina, N Umarani, T Kumar.,”Synthesis and characterization of 2, 5-disubstituted -1,3,4-oxadiazoles as potential inflammatory agents”. Indian J. Chemistry., 2009; 1:72-76.
17. Virginija Jakubkiene, Milda Malvina Burbuliene, Giedrute Mekuskiene, Emilija Udrenaite, Povilas Gaidelis, Povilas Vainilavicius, “Synthesis and anti-inflammatory activity of 5-(6-methyl-2-substituted 4-pyrimidinyl oxymethyl)-1,3,4-oxadiazole-2-thiones and their 3-morpholinomethyl derivatives”. Il Farmaco 2003; 58: 323-/
18. Pelletier JC et al., Preparation of highly substituted gamma lactam FSH receptor agonists, Bioorg. Med. Chem 2005; 13: 5986-5 995.
19. “OECD guidelines – 423” for testing of chemicals, 2001, pp 1-14.

20. Garrat DC. The Quantitative Analysis of Drugs, Japan, Chapman and Hall, 1964, 456.
21. Fine Needle Aspiration Biopsy of the Rat Liver: Cytological, Cytochemical and Biochemical methods. Ed by G. Zbinden. Proc of Workshop; Zurich, May 19, 1st edition.
22. Text Book of Pathology, 6th edition. Harsh Mohan, 612-613.
    

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    How to cite this article:

    Bhaumik A and Eswaraiah MC: Synthesis, characterization and evaluation of hepatocytes regenerator potentiality of some novel oxadiazole derivatives followed by molecular docking against NF-KB. Int J Pharm Sci Res 2017; 8(9): 3734-50. doi: 10.13040/IJPSR. 0975-8232.8(9).3734-50.

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