THE IN-VITRO ANTI-DENATURATION EFFECTS INDUCED BY SUBSTITUTED OXY/THIOXY PYRIMIDINE DERIVATIVES IN BOVINE SERUM ALBUMIN IS PROPOSED AS A SCREENING ASSAY FOR THE DETECTION OF ANTI-INFLAMMATORY COMPOUNDS WITHOUT THE USE OF ANIMALS

THE IN-VITRO ANTI-DENATURATION EFFECTS INDUCED BY SUBSTITUTED OXY/THIOXY PYRIMIDINE DERIVATIVES IN BOVINE SERUM ALBUMIN IS PROPOSED AS A SCREENING ASSAY FOR THE DETECTION OF ANTI-INFLAMMATORY COMPOUNDS WITHOUT THE USE OF ANIMALS

Mrityunjay Banerjee ^{* 1} and Sujit Kumar Sahu ²

Institute of Pharmacy & Technology ¹, Salipur, Cuttack - 754202, Orissa, India.

Biju Patnaik University of Technology ², Rourkela -769004, Odisha, India.

ABSTRACT: A series of novel 2-{[6-(2-substituted Phenyl)-2-oxo-1, 2-dihydropyrimidin -4-yl]oxy} benzoic acid (2a-b)/ 2-{[6-(2-substituted Phenyl)-2 thioxo-1, 2-dihydropyrimidin-4-yl]oxy} benzoic acid (2c-d) were synthesized and tested for their in-vitro protein denaturation activity. Compound 2d was found to be promising and was more potent than the Acetylsalicylic acid (NSAID) in the inhibition of Bovine serum albumin denaturation. The purity of the compounds was checked by TLC and elemental analyses. Both analytical and spectral data (1H NMR, IR, and MS) of all the synthesized compounds were in full agreement with the proposed structures.

Oxy/thioxy Pyrimidine, BSA, Protein denaturation activity

INTRODUCTION: Pyrimidine are versatile Nitrogen-containing heterocyclic compounds possessing broad-spectrum of biological activity and oxopyrimidine, and Thioxopyrimidine has proven record of biological activities, which contains two Nitrogen atoms ^{1, 2}. They are known to exhibit pharmacological activities such as analgesic, anti-inflammatory, anti-arrhythmic, anti-parkinsonian, and anticancer activities ^3-7. Acetylsalicylic acid (Aspirin) is a very useful unit in the fields of medicinal and pharmaceutical chemistry and has been reported to exhibit a variety of biological activities. Chalcones and their analogies having α, β- Unsaturated carbonyl system are very versatile substrates for the evolution of

various reactions and physiologically active compounds chalcones display a large number of different biological activities, such as antibacterial, antifungal, anticancer, anti-inflammatory, anal-gesic, antiviral, antimalarial, antipyretic and cytotoxic activities ^{8, 9}.

The reaction of the urea/thiourea with the different chalcones of acetylsalicylic acid derivative resulting oxo / thioxy pyrimidine ring. In view of the pharmacological profiles of these two chemical moieties as described above, we considered it interesting to synthesized two chemically different but pharmacologically compatible molecules with the aim of obtaining some novel heterocyclic systems with potentially enhanced biological properties. In the present investigation a new series of novel 2-{[6-(2-substituted Phenyl)-2oxo-1, 2-dihydropyrimidin-4-yl]oxy} benzoic acid (2a-b)/ 2-{[6- (2-substituted Phenyl)-2-thioxo-1, 2-dihydro-pyrimidin-4-yl]oxy} benzoic acid (2c-d) were synthesized and evaluated for their in-vitro protein denaturation activity ¹⁰.

SCHEME 1

MATERIALS AND METHODS: Melting points were determined on a Tempstar apparatus and are uncorrected. Infrared spectra were recorded on a Jasco (410) FT-infrared spectrophotometer, measured as KBr disks. 1H NMR were recorded on a Bruker DPX-300 MHz spectrometer in deuteriochloroform with trimethylsilane as internal standard (chemical shift in ppm). The mass spectral data were obtained with a Perkin-Elmer Hitachi RMU-6L MS-30 spectrometer at 70 eV and a 90 °C inlet temperature. Purity of all the compounds was checked on silica gel plates and spots were located in iodine vapours. Elemental analysis was performed on EURO EA (Italy) analyzer and the results were within 0.3% of calculated values. Materials that were used in this study consists of acetylsalicylic acid (C₉H₈O₄), ethanol (C₂H₆O), benzaldehyde (C₇H₆0₂), sodium hydroxide (NaOH), bovine serum albumin, hydrochloric acid (HCl) and urea (CON₂H₄) (Merck).

General Method for the Preparation of 2-{-3-phenylprop-2-enoyloxy} benzoic acid (1a): Equimolar quantity of acetylsalicylic acid (0.01 m) and benzaldehyde (0.01 m) were added in ethanol. 10 ml of 2% sodium hydroxide solution were added dropwise with constant stirring in a magnetic stirrer over a period of 30 min. The reaction mixture was stirred for 17 h and then refluxed for 6 h. then the reaction mixture was poured into ice cold water. The separated solid was filtered, washed with aqueous ethanol and recrystallized from ethanol: water (50:50). Compound 1b was prepared similarly.

2-{-3-phenylprop-2-enoyloxy} benzoic acid (1a): IR (KBr): 3076 (Ar-CH), 1732 (C=O), 1670 (C=O), 1609 (C=C), MS: (m/z) 252(M+); 1H NMR (CDCl₃) δ: 11.69 (s, 1H, COOH), 7.61(d, CH, CH=CH), 7.68 (d, CH, CH=CH), 8.06 (m, 9H, ArH), 6.02-7.58 (m, 4H, ArH);

2-[3-(2-hydroxyphenyl)prop-2-enoyl]benzoic acid  (1b): IR (KBr): 3470 (OH), 3072(Ar-CH), 1730 (C=O), 1672 (C=O), 1602 (C=C), MS: (m/z) 268(M+); 1H NMR (CDCl₃) δ: 11.68 (s, 1H, COOH), 7.66 (d, 1H, CH=CH), 7.74 (d, 1H, CH=CH), 8.06 (m, 9H, ArH), 6.02-7.58 (m, 4H, ArH), 5.69 (s, IH, OH).

General Method for the Preparation of 2-{[6-2-hydroxyphenyl) -2-oxo-1, 2-dihydropyrymidin-4-yl] oxy} benzoic acid (2a): Equimolors quantity of 1a (0.1 M) and urea (0.1 M) were mixed in ethanol. The reaction mixture was refluxed for 4 to 5 h. Then the reaction mixture was poured into ice cold water. The separated solid was filtered, washed with aqueous ethanol, and recrystallized from ethanol: water (50:50), Compound 2b was prepared similarly.

2-(2-oxo- 6- phenyl-1,2,5,6-tetrahydropyrimidin-4-yl)benzoic acid: (2a)  IR (KBr): 3346 (NH), 3075 (Ar-CH), 1731 (C=O), 1682 (C=O), 1607 (C=N), MS: (m/z) 310(M+); 1H NMR (CDCl₃) δ: 11.72 (s, 1H, COOH), 7.52 (d, 1H, NH), 5.23 (q, 1H, CH), 3.24(d, 2H, CH₂), 6.02-7.49 (m, 4H, ArH).

2-[6-(2- hydroxyphenyl) -2-oxo-1, 2, 5, 6-tetrahydropyrimidin-4-yl] benzoic acid  (2b): IR (KBr): 3471 (OH), 3344(NH), 3076(Ar-CH), 1732 (C=O), 1682 (C=O), 1610 (C=N), MS: (m/z) 326 (M+); 1H NMR (CDCl₃) δ: 11.71 (s, 1H, COOH), 7.51 (d, 1H, NH), 5.19(q,1H,CH), 3.22 (d, 2H, CH₂), 6.02-7.47 (m, 4H, ArH), 5.66 (s, IH, OH).

General method for the preparation of 2-(6-phenyl-2-thioxo-1, 2, 5, 6-tetrahydropyrimidin-4-yl) benzoic acid (2c): Equimolors quantity of 1a (0.1 M) and urea (0.1 M) were mixed in ethanol. The reaction mixture was refluxed for 4 to 5 h. Then the reaction mixture was poured into ice cold water. The separated solid was filtered, washed with aqueous ethanol and recrystallized from ethanol: water (50:50), Compound 2d was prepared similarly.

2-{[6-2- hydroxyphenyl) -2-thioxo-1, 2-dihydropyrymidin-4-yl] oxy} benzoic acid (2c): IR (KBr): 3345(NH), 3075 (Ar-CH), 1730 (C=O), 1682 (C=O), 1609 (C=N), MS: (m/z) 326(M+); 1H NMR (CDCl₃) δ: 11.74 (s, 1 H, COOH), 7.57 (d, 1H, NH), 5.29 (q, 1H, CH), 3.28 (d, 2H, CH₂), 6.08-7.47 (m, 4H, ArH).

2-[6-(2- hydroxyphenyl)-2-thioxo-1, 2, 5, 6-tetrahydropyrimidin-4-yl]benzoic acid (2d):  IR (KBr): 3473 (OH), 3342(NH), 3076(Ar-CH), 1733 (C=O), 1684 (C=O), 1612 (C=N), MS: (m/z) 342 (M+); 1H NMR (CDCl₃) δ: 11.72 (s, 1H, COOH), 7.56 (d, 1H, NH), 5.31(q,1H,CH), 3.32 (d, 2H, CH₂), 6.08-7.47 (m, 4H, ArH), 5.63 (s, IH, OH);

TABLE 1: PHYSICAL DATA OF SYNTHESIZED COMPOUNDS 1A-B & 2A-D  

Inhibition of Protein Denaturation: The reaction mixtures (0.5 ml) consisted of 0.45 ml bovine serum albumin (5% aqueous solution) and 0.05 ml of test compound (100 and 250 mg/ml of final volume). pH was adjusted at 6.3 using a small amount of IN HCl. The samples were incubated at 37 ºC for 3 min. After cooling the samples, 2.5 ml phosphate buffer saline (pH 6.3) was added to each tube. Turbidity was measured spectrophoto-metrically at 660 nm. For control tests, 0.05ml distilled water was used instead of synthesized product control tests that lacked bovine serum albumin. The percentage inhibition of protein denaturation was calculated as follows

100 - (O.D. of test – O.D. of product control) / O.D. of control × 100

TABLE 2: IN-VITRO ANTI-INFLAMMATORY SCREENING OF SYNTHESIZED COMPOUNDS BY BOVINE SERUM ALBUMIN DENATURATION

*Average of Three readings

RESULTS AND DISCUSSION: 2-{-3-phenylprop-2-enoyloxy} benzoic acid (1a) and 2-[3-(2-hydroxyphenyl) prop-2-enoyl] benzoic acid 1b were prepared by reacting with aspirin and substituted benzaldehyde. Compounds 1a,b were condensed with urea or thiourea in ethanol to afford the corresponding pyrimidine derivatives 2a, 2b or 2c, 2d in 69-71% yields Table 1. All the synthesized compounds were characterized by their elemental analysis, FT-IR, 1H NMR, and mass spectroscopy. Various 2-{[6-(2- substituted Phenyl) -2oxo-1, 2-dihydropyrimidin-4-yl] oxy} benzoic acid (2a-b)/ 2-{[6-(2-substituted Phenyl)-2oxo-1, 2-dihydropyrimidin-4-yl]oxy} benzoic acid (2c-d) were tested for their inhibition of protein denaturation activity by BSA assay method ⁸ using Bovine Serum Albumin.

Among the 4 compounds synthesized, two compounds exhibited significant activity. Hence, we can conclude that the size of hydroxyl substituents at the position of thioxy pyrimidine moiety is important for in-vitro anti-inflammatory activity. Among the newer derivatives, compound 2d showed promising activity in the test. It is conceivable that thioxy derivatives showing in-vitro Protein denaturation activity can be further modified to achieve NSAID agents with antiarthritis activity.

CONCLUSION: All the compounds have been screened for their in-vitro anti-inflammatory activity, advantage of present proposed research work was cost effective as compared to the in-vivo method.

Generally, it can be concluded that 2-[6-(2-hydroxyphenyl)- 2- thioxo-1, 2, 5, 6-tetrahydro-pyrimidin-4-yl] benzoic acid (2d) represent a new distinct classes of anti-inflammatory agents with antiarthritis activity. Compound 2d was found to be promising compared to the Acetylsalicylic acid (NSAID) in the inhibition of Bovine serum albumin denaturation. Further investigations with appropriate structural modifications of title compounds may result in therapeutically useful products.

All this is supremely encouraging for further research in this arena of anti-inflammatory drug discovery.

ACKNOWLEDGEMENT: The authors are grateful to the authorities of Department of Pharmaceutical Chemistry, Institute of Pharmacy and Technology, Salipur, Cuttack, Orissa, India for providing the necessary facility to carryout this research work.

CONFLICTS OF INTEREST: Nil

REFERENCES:

1. Latif NA, Saeed MM, Ahmed NS, Batran RZ and El-Mouhty NRA: Synthesis of some pyridine, pyrimidine and cyclohexenone derivatives as antibacterial agents. International Journal of Innovative Research in Science, Engineering and Technology 2014; 3(1): 8517-29.
2. Tomma JH, Khazaal MS and Al-Dujaili AH: Synthesis and characterization of novel Schiff bases containing pyrimidine unit. Arabian J of Chem 2014; 7(1): 157-163.
3. Baumann M and Baxendale IR: An overview of the synthetic routes to the best selling drugs containing 6-membered heterocycles. Beilstein J Org Chem 2013; 9: 2265-19.
4. Nayak SG and Poojary B: Synthesis of novel Schiff bases containing aryl pyrimidines as promising antibacterial agents. Heliyon 2019; 5(8): 1-7.
5. Hammam AG, Fahmy AFM, Amr AE and Mohamed AM: Synthesis of novel tricyclic heterocyclic compounds as potential anticancer agents using chromanone and thio-chromanoneas synthons. Ind J Chem 2003; 42B: 1985-93.
6. Sharma V, Chitranshi N and Agarwal AK: Significance and biological importance of pyrimidine in the microbial world. International J of Medicinal Chemistry 2014; 1-31.
7. Elavarasan M, Thendral MT and Syed SS: Synthesis, characterization and antimicrobial activity of some new chalcones. IJPSR 2018; 9(5): 1969-73.
8. Tiwari B, Pratapwar AS, Tapas AR, Butle SR and Vatkar BS: Synthesis and Antimicrobial Activity of Some Chalcone Derivatives. IJCRGG 2010; 2(1): 499-03.
9. Xie D, Xie Y, Ding Y, Wu J and Hu D: Synthesis of Chiral Chalcone Derivatives Catalyzed by the Chiral Cinchona Alkaloid Squaramide. Molecules 2014; 19: 19491-00.
10. Dasgupta S, Fishman MA, Castro LM, Tashima AK, Ferro ES and Fricker LD: Effect of Protein Denaturation and Enzyme Inhibitors on Proteasomal-Mediated Production of Peptides in Human Embryonic Kidney Cells. Biomolecules 2019; 207(9): 1-18.
    

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    How to cite this article:

    Banerjee M and Sahu SK: The in-vitro anti-denaturation effects induced by substituted oxy/thioxy pyrimidine derivatives in bovine serum albumin is proposed as a screening assay for the detection of anti-inflammatory compounds without the use of animals. Int J Pharm Sci & Res 2020; 11(4): 1885-88. doi: 10.13040/IJPSR.0975-8232.11(4).1885-88.

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