Design, Molecular docking, Synthesis and Biological evaluation of

5, 7 dimethyl pyrido (2, 3-d) pyrimidin-4-one and 4,5 dihydro pyrazolo (3, 4-d) pyrimidines for cytotoxic activity

 

M. Sathish Kumar¹, M. Vijey Aanandhi²*

¹Research Scholar, Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences, Vels Institute of Science, Technology and Advanced Studies,

VISTAS, Pallavaram, Chennai - 600117 Tamilnadu, India

²Department of Pharmaceutical Chemistry and Analysis, School of Pharmaceutical Sciences,

Vels Institute of Science, Technology and Advanced Studies,

VISTAS, Pallavaram, Chennai - 600117 Tamilnadu, India

 

ABSTRACT:

The fused pyrimidine derivatives are potent tyrosine kinase and thymidylate synthase inhibitors. The compound 3-(4-sulphonyl amino)-2-methyl thio-6-phenyl azo-5, 7-dimethyl pyrido(2,3-d)pyrimidin-4-one was synthesized from Ethyl 2-amino-4,6-dimethylpyridine-3-carboxylate, benzene diazonium chloride, benzene sulphonyl amino isothiocyanate in subsequent reactions. 1-(1, 3-benzothiazol-2-yl)-3-methyl-4-phenyl-1H-pyrazolo[3,4-d]pyrimidines were synthesized from 1, 3-benzothiazole, 2-thiol, Hydrazine Hydrate, 2-hydrazinyl-1, 3-benzothiazole and aldehydes in subsequent reactions. Twenty-five derivatives pyrimidine scaffolds were designed and performed molecular docking studies for the ability to inhibit the target protein using molecular docking simulation, selective compounds were synthesized and characterized by spectral methods.  All the synthesized compounds evaluated for their antioxidant activity and MTT assay exhibited compounds 13c, 13e and 14d can be potential anticancer candidates against MCF-7, Hep G2 and Hela cell lines respectively. Based on all the studies conclude that good agreement was observed between the top-ranked docking scores and top experimental inhibitors when compared with standards ascorbic acid and imatinib. Hence, the compounds could be considered as new anticancer hits for further lead optimization.

 

 

 

INTRODUCTION:

Pyrimidines have a long and distinguished history extending from the days of their discovery as important constituents of nucleic acids to their current use in the chemotherapy. pyrimidines and its fused pyrimidines are the class of heterocyclic scaffolds that demonstrate a wide range of pharmacological effects such as anti-inflammatory^1,2, antimicrobial^3-5, anti-HIV^6,7, anti-malarial^8-10, anticancer activities^11-20, antimycobacterial^21-25, antiangiogenic^26-27 etc. 

 

However, the pyridopyrimidine are the least attention so far because of its difficulty and expensiveness. Pyrido[3,2-d]pyrimidines reported as  tyrosine kinase inhibitors^28-30, anticonvulsant³¹, antifungal³², anxiolytic³³, antioxidant^34,35, antidepressant³⁶, etc. The tyrosine kinase and thymidylate synthases have been recognized as new molecular targets for treatment of cancer. Cell division and differention is important phenomena in cells that regulate by signal transduction associated with receptor tyrosine kinases. Imatinib and dasatinib are new anticancer drugs with mechanism related to the inhibition of tyrosine kinases. Some heterocyclic compounds such as fused pyrido pyrimidine and pyarazolo pyrimidines are very attractive research area in chemotherapy. Chemistry of pyrazolo[3,4-d]pyrimidine derivatives and pyrazolo[3,4-d]pyrimidine derivatives has received great attention due to their structural similarity with nucleotides and hence such fused scaffolds exhibit promising anticancer activity. Nasser S.M. Ismail et al.³⁷, Pyrazolopyrimidines were explored towards various kinases as cytotoxic agents as well as structural requirements for kinase affinity through in silico studies. P. Shanmugasundaram et al.³⁸ pyrimidine-carboxylates tested for antitumor studies by MTT assay and reported the compounds better anticancer activity profile. Some popular medications in the market like 5-fluorouracil, tigafur, thiaguanine, imatinib and dasatinib are fused pyrimidine derivatives. Potential of anticancer activity of the structure is most interesting area that has been encouraged researchers to focus on it. Hence, the large number of molecules was designed, investigations were done by molecular docking studies on two types of cancer cellular kinases including Thymidylate Synthase and Tyrosine Kinase and the binding modes and interactions were compared with standards imatinib and dasatinib and based on the reports of docking studies some selective molecules were synthesized, characterized by spectral analysis and evaluated for antioxidant and anticancer activities.                                   

 

MATERIALS AND METHODS:

For present work databases like Protein Data Bank (PDB), Drug bank, and Molecular docking software’s like GLIDE 5.0 module implemented in the Schrodinger. As per mentioned schematic representations the fused pyridopyrimidines and pyrazolopyrimidine derivatives were designed using Chem sketch software and generated their mol file followed subsequent generations of their 3D-Strucures by using tool Web lab lite viewer program molecule format convert into PDB. Appropriate force field was applied to them and the optimization was carried using glide (https://www.schrodinger.com/ glide)³⁹.

 

Docking studies:

The crystal structure of Thymidylate Synthase (TS) and Tyrosine Kinase (TK) were selected as a ligand target as PDB ID: 1TSM and 2SRC has been retrieved from RCSB protein Data Bank. Then PDB file was converted into PDBQT file by using Auto Dock Tools (ADT) version 1.5.6 from the Scripps Research Institute. Drug-likeness properties of designed molecules were analyzed by Lipinski^’s rule of five and bonding interactions on the basis of scoring functions including Hydrogen Bonding Donor (HBD), Hydrogen Bonding Acceptor (HBA), Lipophilicity (Log p) value is significant property to predict the bioavailability have ideal value ≤ 5 and good Glide Score in bonding with more than one amino residues except few molecules and they occupy the active pockets of protein express in Kcal per mole.

 

Table 1: Binding free energies of the compounds after docking to the active site of Thymidylate Synthase and Tyrosine Kinases

 

 

Fig. 1: Structures of two clinically used tyrosine kinase inhibitors A: Imatinib   B: dasatinib

 

Fig. 2: Synthetic Schemes

 

All the required chemicals were obtained from Aldrich-Chemicals and SD-Fine Chemicals. All the solvents used in the synthesis were laboratory grade. Each reaction was monitored by TLC using appropriate solvent system. Precoated TLC plates (0.25mm silica gel) obtained from E. merck. All the synthesized compounds were purified by recrystallization.  All melting points were determined in open capillary tubes and are uncorrected. All FT-IR spectrum were recorded on Thermo Nicolet Nexus 4700 FT-IR instrument KBr disc method. All the ¹HNMR spectrum was recorded on Advance 300 MHz Bruker UX-NMR instrument and the samples were made DMSO-d₆ using tetra methyl silane (TMS) as internal standard. The entire mass spectrum was recorded on Thermo Finnigan LCQ Ion Trap instrument and they were reported in m/z as molecular ion peak.

 

RESULTS AND DISCUSSION:

Synthesis and Spectral data of the Synthesized Compounds

3-(4-sulphonyl amino)-2-methyl thio-6-phenyl azo-5, 7-dimethyl pyrido(2,3-d)pyrimidin-4-one (5e):

A mixture of Ethyl 2-amino-4, 6-dimethylpyridine-3-carboxylate (1) (0.1mol) with benzene diazonium chloride (0.3mol) was refluxed for 6 hours  in butanol (20ml). The reaction mixture was left to cool; the precipitate was collected and recrystallized from ethanol. A mixture of compound Ethyl 2-amino-4,6-dimethyl-5-[phenyldiazenyl]pyridine-3-carboxylate (2) with benzene sulphonyl amino isothiocyanate in ethanol at reflux for 4-5 hours including addition of Conc. HCl (3-4ml) intermittently. The reaction mixture was left to cool; the precipitate was collected and recrystallized from ethanol. A mixture of compound 5, 7-dimethyl-6-[phenyldiazenyl]-2-thioxo-2, 3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one (4) with dimethyl sulfate in ethanolic sodium hydroxide solution (4gm in 20ml) at reflux for 7-8 hours. The reaction mixture was left to cool; allow the mixture to stand for 24 hours at room temperature. Filter the brown color precipitate and wash with water and recrystallized from ethanol.

 

Light brown crystalline solid; yield 51%; m. p. 255-65°C; IR (KBr): 3024, 1599, 1680, 2132, 2924, 3381, 1370 cm^-1, ¹H-NMR (DMSO-d₆): δ 2.31 (3H, s), 3.78 (3H, s), 5.92 (1H, s), 6.84 (1H, d), 7.14 (2H, d), 7.21-7.36 (4H, m), 7.54-7.61 (4H, m), 8.15 (1H, d).; HRMS: m/z [M+H⁺] calcd for C₂₂H₂₀N₆O₃S₂: 480.642. Found: 480.254; Composition calcd: C (54.98%) H (4.19%) N (17.49%) O (9.99%) S (13.34%); found: C (54.44%) H (4.53%) N (17.98%) O (9.01%) S (13.23%)

 

Synthesis of 1-(1, 3-benzothiazol-2-yl)-3-methyl-4-phenyl-1H-pyrazolo[3,4-d]pyrimidine (9b):

A mixture of 1, 3-benzothiazole, 2-thiol (6) (0.1mol) and Hydrazine Hydrate (0.1mol) in 40ml water and refluxed for 3 hours at 50-60^oc. Cool the reaction mixture. Filter the product to precipitate out. Keep the solution in bath for 24 hours. Filter the white product. Wash with water. A mixture of compound 2-hydrazinyl-1, 3-benzothiazole (7) (0.1mol) with ethyl aceto acetate (12ml) in alkali ethanolic solution about 20ml, refluxed for 4-5 hours. The reaction mixture was left to cool, the precipitate was collected and recrystallized from ethanol gives the compound 1-(1, 3-benzothiazol-2-yl)-3-methyl-1, 4-dihydro-5H-pyrazol-5-one (8). A mixture of compound 8 (0.2mol) with benzaldehyde (0.2mol) and urea (0.5mol) in ethanol about 30ml, refluxed for 5-6 hours. Cool the reaction mixture, filter the product and recrystallize the product using ethanol.

 

White crystalline solid; yield 64%; m. p. 225-35°C; IR (KBr): 3048, 2970, 1627, 1599, 752 cm^-1; ¹H-NMR (DMSO-d6): δ 2.47 (3H, s), 6.62-6.71 (3H, d), 6.97 (1H, d), 7.13 (1H, d), 7.40 (2H, d), 7.63 (1H, t), 7.98 (2H, m), 9.15 (1H, s); HRMS: m/z  [M+H⁺] calcd for C₁₉H₁₃N₅S:343. 435. Found: 343.432; Composition calcd: C (66.45%) H (3.82%) N (20.39%) S (9.34%); Found: C (66.15%) H (3.25%) N (20.56%) S (9.87%)

 

Synthesis of 1-(1, 3-benzothiazol-2-yl)-3-methyl-4-hydroxy-phenyl-1H-pyrazolo [3, 4-d] pyrimidine (9g):

A mixture of compound (1, 3-benzothiazol-2-yl)-3-methyl-1,4-dihydro-5H-pyrazol-5-one (8) (0.2mol) with 4-hydroxy benzaldehyde (0.2mol) and urea in ethanol about 30ml, refluxed for 5-6 hours. Cool the reaction mixture. Filter the product and wash at the pump. Recrystallize the product using ethanol.

 

White crystalline solid; yield 57%;  m. p. 235-45°C; IR (KBr): 3044, 2972, 1625 , 1598, 3245, 750 cm^-1. ¹H-NMR (DMSO-d₆): ¹H NMR: δ 2.42 (3H, s), 6.59 (1H, s), 6.62-6.71 (2H, d), 6.97 (1H, d), 7.13 (1H, d), 7.30 (2H, d), 8.05 (2H, d), 9.11 (1H, s).; HRMS: m/z [M+H⁺] calculated for C₁₉H₁₃N₅OS; 359.756. Found: 359.234; Composition calcd: C (63.49%) H (3.65%) N 19.49%) O (4.45%) S (8.92%); found: C (63.15%) H (3.54%) N (19.25%) O (4.58%) S (8.12%)

 

1-{4-[(5-methoxypyridin-2-yl)amino]phenyl}-3-methyl-4-phenyl-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-6-ol(13a):

A mixture of 3-methyl 2-choro pyridine (10) (0.5mol) with 1-(4-amino)-3-methyl pyrazole-5-one (11) (0.5mol) in 40ml ethanol reflux for 5 hours. Cool the reaction mixture and allow standing for 24 hours in refrigerator. Filter the product and wash at the pump gives the compound 12a. A mixture of compound 12a (0.2mol) with urea (0.2mol) and benzaldehyde (0.3mol) in ethanol about 40ml under reflux for 6-7 hours. Cool the reaction mixtute to room temperature, add about 50ml of ice cold water. Filter the precipitate, wash with cold water and recrystallize using ethanol.

 

Light brown crystalline solid; yield 62%; m. p. 260-70°C; IR (KBr): 3378, 3232, 1733, 1576, 1350, 754 cm^-1; ¹H NMR (300 MHz, CDCl₃): ¹H NMR: δ 2.13 (3H, s), 2.29 (3H, s), 5.69 (1H, s), 6.40 (1H, d), 6.87 (2H, m), 7.23-7.35 (3H, d), 7.55-7.62 (4H, d), 8.22 (1H, d); HRMS: m/z [M+H⁺] calculated for C₂₄H₂₂N₆O₂: 397.45. Found: 397.40; Composition calcd:  C (70.23%) H (5.40%) N (20.47%) O (3.90%); found: C (70.28%) H (5.18%) N (20.07%) O (3.19%)

 

1-{4-[(5-methoxypyridin-2-yl)amino]phenyl}-3-methyl-4-phenyl-4,5-dihydro-1H-pyrazolo[3,4-d] pyrimidin-6-ol (13b):

A mixture of 3-methoxy 2-choro pyridine (10) (0.5mol) with 1-(4-amino)-3-methyl pyrazole-5-one (11) (0.5mol) in 40ml ethanol reflux for 5 hours. Cool the reaction mixture and allow standing for 24 hours in refrigerator. Filter the product and wash at the pump gives the compound 12b. A mixture of compound 12b (0.2mol) with urea (0.2mol) and benzaldehyde (0.3mol) in ethanol about 40ml under reflux for 6-7 hours. Cool the reaction mixtute to room temperature, add about 50ml of ice cold water. Filter the precipitate, wash with cold water and recrystallize using ethanol.

 

Light yellow crystalline solid; yield 53%; m. p. 255-65°C; IR (KBr): 3474, 3232, 1733, 1576, 759 cm^-1; ¹H NMR (300 MHz, CDCl₃): δ 2.29 (3H, s), 3.78 (3H, s), 5.69 (1H, s), 6.82-6.91 (3H, m), 7.36 (4H, m), 8.15 (1H, d); HRMS: m/z [M+H⁺] calculated for C₂₄H₂₂N₆O₂: 426.255. Found:426.453; Composition calcd:  C(64.11%) H (4.44%) Cl (8.23%) N (19.50%) O (3.71%); found: C (64.25%) H (4.87%) Cl (8.58%) N (19.55%) O (3.74%)

 

1-{4-[(5-chloropyridin-2-yl) amino] phenyl}-3-methyl-4-phenyl-4, 5-dihydro-1H-pyrazolo[3, 4-d]pyrimidin-6-ol (13c):

A mixture of 3-chloro pyridine (10) (0.5mol) with 1-(4-amino)-3-methyl pyrazole-5-one (11) (0.5mol) in 40 ml ethanol reflux for 5 hours. Cool the reaction mixture and allow standing for 24 hours in refrigerator. Filter the product and wash at the pump gives the compound 5-methyl-2-{4-[(5-chloropyridin-2-yl) amino] phenyl}-2, 4-dihydro-3H-pyrazol-3-one (12c).A mixture of compound 12c (0.2mol) with urea (0.2mol) and benzaldehyde (0.3 mol) in ethanol about 40ml under reflux for 6-7 hours. Cool the mixture to room temperature, add about 50 ml of ice cold water, filter the precipitate, wash with cold water and recrystallize using ethanol.

 

Light yellow crystalline solid; yield 63%; m. p. 235-45°C; IR (KBr pellets): 3076, 1447, 1241, 1691, 1158, 867, 735 cm^-1; ¹H NMR(300 MHz, CDCl₃): δ 2.29 (3H, s), 5.69 (1H, s), 6.87 (2H, d), 7.23 (2H, d), 7.23-7.40 (6H, m), 7.60 (2H, m), 8.07 (1H, m), 8.96 (1H, m); HRMS: m/z [M+H⁺] calculated for C₂₃H₁₉ClN₆O: 430.564.found:430.264; Composition calcd: C (64.11%) H (4.44%) Cl (8.23%) N (19.50%) O (3.71%); Found: C (64.87%) H (4.58%) Cl (8.25%) N (19.87%) O (3.14%)

 

1-{4-[(5-hydroxypyridin-2-yl) amino] phenyl}-3-methyl-4-phenyl-4, 5-dihydro-1H-pyrazolo[3, 4-d]pyrimidin-6-ol (13e):

A mixture of 3-hydroxy pyridine (10) (0.5mol) with 1-(4-amino)-3-methyl pyrazole-5-one (11) (0.5mol) in 40 ml ethanol reflux for 5 hours. Cool the reaction mixture and allow standing for 24 hours in refrigerator. Filter the product and wash at the pump gives the compound 5-methyl-2-{4-[(5-hydroxypyridin-2-yl) amino] phenyl}-2,4-dihydro-3H-pyrazol-3-one (12e). A mixture of compound 12e (0.2mol) with urea (0.2mol) and benzaldehyde (0.3 mol) in ethanol about 40ml under reflux for 6-7 hours. Cool the mixtute to room temperature, add about 50ml of ice water. Filter the precipitate, recrystallize using ethanol.

 

White crystalline solid; yield 54%; m. p. 215-25°C; IR (KBr): 3368, 1691,1470, 1241,1108, 886, 708 cm^-1; ¹H NMR (300 MHz, CDCl₃): δ 2.29 (3H, s), 5.69 (1H, s), 6.87 (1H, m), 7.11 (2H, m), 7.50-7.63 (7H, m), 7.60 (1H, m), 7.99 (1H, m); HRMS: m/z [M+H⁺] calculated for C₂₃H₂₀N₆O₂: 412.305.found:413.0; Composition calcd: C (66.98%) H (4.89%) N (20.38%) O (7.76%); found: C(66.78%) H (4.58%) N (20.21%) O (7.78%)

 

1-{4-[(5-carboxypyridin-2-yl) amino] phenyl}-3-methyl-4-phenyl-4, 5-dihydro-1H-pyrazolo [3, 4-d] pyrimidin-6-ol (13f):

A mixture of 3-pyridine carboxylic acid (10) (0.5mol) with 1-(4-amino)-3-methyl pyrazole-5-one (11) (0.5mol) in 40 ml ethanol reflux for 5 hours. Cool the reaction mixture and allow standing for 24 hours in refrigerator. Filter the product and wash at the pump gives the compound 5-methyl-2-{4-[(5-corboxypyridin-2-yl) amino] phenyl}-2,4-dihydro-3H-pyrazol-3-one  (12f). A mixture of compound 12f (0.2mol) with urea (0.2mol) and benzaldehyde (0.3mol) in ethanol about 40ml under reflux for 6-7 hours. Cool mixture to room temperature, add about 50ml of ice water, Filter the precipitate and recrystallize the product using ethanol.

 

Light yellow crystalline solid; yield 41%; m. p. 245-50°C; IR (KBr): 3474, 1680, 3241,1733, 856, 765, 543 cm^-1; ¹H NMR (300 MHz, CDCl₃): δ 2.29 (3H, s), 5.69 (1H, s), 6.79-6.91 (3H, m), 7.23-7.35 (3H, m), 7.53-7.63 (4H, m), 7.79 (1H, d), 8.56 (1H, d); HRMS: m/z [M+H⁺] calculated for C₂₄H₂₀N₆O₃: 440.432. Found: 440.765; Composition calcd: C (63.14%) H (4.42%) N (18.41%) O (7.01%) S (7.02%); found: C (63.74%) H (4.14%) N (18.85%) O (7.58%) S (7.73%)

 

3-methyl-4-phenyl-1-[4-(5-methyl-pyridin-2-yl-amino) phenyl]-4, 5-dihydro-1H-pyrazolo [3, 4-d] pyrimidine-6-thiol (14a):

A mixture of 4-methoxy 2-chloropyridine (10) (0.5mol) with 1-(4-amino)-3-methyl pyrazole-5-one (11) (0.5mol) in 40ml ethanol reflux for 5 hours. Cool the reaction mixture and allow standing for 24 hours in refrigerator. Filter the product and wash at the pump gives the compound 12a which 0.5mol with urea (0.5mol) and benzaldehyde (0.3mol) in ethanol about 40ml under reflux for 6-7 hours. Cool mixture to room temperature, add about 50ml of ice water, Filter the precipitate and recrystallize the product using ethanol.

 

Light brown solid; yield 65%; m. p. 195-205°C; IR (KBr): 3328, 3252, 3214, 2585, 2124, 1524,  845, 774 cm^-1; ¹H NMR: δ 2.13 (3H, s), 2.31 (3H, s), 5.92 (1H, s), 6.40 (1H, d), 7.14 (2H, d), 7.21-7.36 (3H, m), 7.54-7.62 (4H, m), 7.86 (1H, d); HRMS: m/z [M+H⁺] calculated for C₂₄H₂₂N₆S: 426.54. Found: 426.14; Composition calcd: C (65.28%) H (4.54%) N (18.57%) O (5.25%) S (6.25%); found: C (65.08%) H (4.12%) N (18.07%) O (6.00%) S (6.54%)

 

3-methyl-4-phenyl-1-[4-(5-methoxy-pyridin-2-yl-amino)phenyl]-4,5-dihydro-1H-pyrazolo [3, 4-d] pyrimidine-6-thiol (14b):

A mixture of 4-methoxy 2-chloropyridine (10) (0.5mol) with 1-(4-amino)-3-methyl pyrazole-5-one (11) (0.5mol) in 40 ml ethanol reflux for 5 hours. Cool the reaction mixture and allow standing for 24 hours in refrigerator. Filter the product and wash at the pump gives the compound 12b which 0.5mol with urea (0.5mol) and benzaldehyde (0.3mol) in ethanol about 40 ml under reflux for 6-7 hours. Cool mixture to room temperature, add about 50ml of ice water, Filter the precipitate and recrystallize the product using ethanol.

 

Yellow crystalline solid; yield 55%; m. p. 200-210°C; IR (KBr): 3385, 3225, 3208, 2625, 2150, 1548, 1733, 1265, 1048, 865, 754 cm^-1; ¹H NMR: δ 2.31 (3H, s), 3.78 (3H, s), 5.92 (1H, s), 6.84 (1H, d), 7.14 (2H, d), 7.21-7.36 (4H, m), 7.54-7.61 (4H, m),  8.15 (1H, d); HRMS: m/z [M+H⁺] calculated for C₂₄H₂₂N₆OS: 442.45. Found: 442.28; Composition calcd: C (65.14%) H (5.01%) N (18.99%) O (3.62%) S (7.25%); found: C (65.14%) H (5.01%) N (18.99%) O (3.62%) S (7.25%)

 

3-methyl-4-phenyl-1-[4-(5-(4-nitrophenyl)-pyridin-2-yl-amino)phenyl]-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidine-6-thiol (14d):

A mixture of 4-(4-nitrophenyl)-2-chloropyridine (10) (0.5mol) with 1-(4-amino)-3-methyl pyrazole-5-one (11) (0.5mol) in 40 ml ethanol reflux for 5 hours. Cool the reaction mixture and allow standing for 24 hours in refrigerator. Filter the product and wash at the pump gives the compound 12d which 0.5mol with urea (0.5mol) and benzaldehyde (0.3mol) in ethanol about 40 ml under reflux for 6-7 hours. Cool mixture to room temperature, add about 50ml of ice water, Filter the precipitate and recrystallize the product using ethanol.

brown crystalline solid; yield 62%; m. p. 205-215°C; IR (KBr): 3324, 3225, 2622, 2150, 1548, 1733, 865, 754, 668 cm^-1; ¹H NMR:δ 2.31 (3H, s), 5.92 (1H, s), 7.14 (2H, d), 7.18-7.35 (4H, m), 7.36-7.47 (3H, d), 7.50 (2H, d), 7.61 (2H, d), 7.70 (2H, m), 7.96 (1H, d), 8.97 (1H, d); HRMS: m/z [M+H⁺] calculated for C₂₉H₂₃N₇O₂S: 533.24. Found: 533.18; Composition calcd: C (65.28%) H (4.34%) N (18.37%) O (6.00%) S (6.01%); found: C (65.05%) H (4.15%) N (18.07%) O (6.00%) S (5.54%)

 

3-methyl-4-phenyl-1-[4-(5-carboxyl-pyridin-2-yl-amino)phenyl]-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidine-6-thiol (14f):

A mixture of 2-chloropyridine 4-carboxylic acid (10) (0.5mol) with 1-(4-amino)-3-methyl pyrazole-5-one (11) (0.5mol) in 40 ml ethanol reflux for 5 hours. Cool the reaction mixture and allow standing for 24 hours in refrigerator. Filter the product and wash at the pump gives the compound 12f which 0.5mol with urea (0.5mol) and benzaldehyde (0.3 mol) in ethanol about 40 ml under reflux for 6-7 hours. Cool mixture to room temperature, add about 50 ml of ice cold water, Filter the precipitate and recrystallize the product using ethanol.

 

White crystalline solid; yield 49%; m. p. 215-225°C; IR (KBr): 3358, 3241, 2610, 2150, 1578, 1733, 858, 755, 643 cm^-1; ¹H NMR: δ 2.31 (3H, s), 5.92 (1H, s), 6.82 (1H, d), 7.14 (2H, m), 7.21-7.35 (3H, m), 7.50 (2H, m), 7.79 (1H, d), 8.56 (1H, d); HRMS: m/z [M+H⁺] calculated for C₂₄H₂₀N₆O₂S: 456.98. Found: 456.25; Composition calcd: C (63.14%) H (4.42%) N (18.41%) O (7.01%) S (7.02%); found: C (63.25%) H (4.28%) N (18.01%) O (7.01%) S (6.58%)

 

Figure 3: The molecular interactions of some fused pyrimidines with thymidylate synthase (TS)

 

DPPH assay:

In this assay, diphenyl picrylhydrazyl (purple color) reacts with antioxidant and is reduced into diphenyl picrylhydrazine (yellow). The intensity of the color is inversely proportional to the capacity of the antioxidant present in the medium to give hydrogen or electrons. The antiradical activity was evaluated spectrophotometrically by following the reduction of the radical at 517nm. 0.2mM solution of DPPH in methanol was prepared and 100μl of this solution was added to title compounds (10-50μg/ml). After 30 minutes, absorbance was measured at 517nm using ascorbic acid as standard⁴⁰.

 

In order to prepare 0.2mM of DPPH solution 7.8mg dissolved in 100ml of methanol (95%) in dark room. Ascorbic acid was used as the standard at the concentration range of 0.156-1.25µg/ml. To Prepare different concentrations of ascorbic acid (AA) 1mg in 1 ml methanol (stock solution) then prepare a series of dilutions from the stock solution to give 0.156, 0.312, 0.625 and 1.25µg/ml.  Leave for 30 min then read the absorbance at 517nm, plot the absorbance versus concentration and calculate IC₅₀.

 

To prepare test substances 1mg of synthesized compound was dissolved in 10ml of methanol in order to prepare 100µg/ml solutions and then serial dilutions were performed to prepare required concentrations. 3ml of DPPH Solution was used a negative control. Briefly, 0.1ml of different concentrations of methanolic solution of standard (0.156-1.25µg/ml) and test compounds (0.156, 0.312, 0.625 and 1.25µg/ml) were added to 2ml of DPPH methanol solution (60mM). The mixture was shaken vigorously and allowed to react at room temperature and in darkness for 5 hrs. The absorbance of the resulting solution was measured at 517nm using a UV/Vis spectrophotometer after 5 hours incubation. Scavenging of DPPH free radicals was calculated as:

 

DPPH scavenging activity (%) = [(Ac–At) / Ac] × 100 where, Ac is the absorbance of the control tube

(Containing all reagents except the test compound), and At is the absorbance of the test tube.

 

Table 2: Antioxidant activity by DPPH method

 

Hydrogen Peroxide Radical Scavenging Assay:

Oxidase enzymes play key role in the generation of hydrogen peroxide in vivo and generated through the reduction product called hydroxyl radical (OH•) hydrogen peroxide is scavenged. Here hydrogen peroxide is scavenged by the test sample, which is the basis of this method. The solution of hydrogen peroxide (40 mM) was prepared by using phosphate buffer pH 7.4; using ascorbic acid as standard and its concentrations as well as test concentrations were made as 0.156, 0.312, 0.625 and 1.25µg/ml. To hydrogen peroxide solution (0.6ml) test samples were added and its concentrations determined by measuring the absorbance at 560nm using UV spectrophotometer against a blank solution phosphate buffer without hydrogen peroxide⁴¹.

 

The percentage of hydrogen peroxide scavenging by the extract and standard compound was calculated using the given formula:

 

Percentage scavenged [H₂O₂] = 1-Abs (standard)/Abs (control) x100

 

Where, Abs control was the absorbance of the control (without substance to be tested) at 560nm; Abs sample was the absorbance in the presence of the test compounds at 560nm. The experiment was repeated in triplicate.

Table 3: Antioxidant activity by Hydrogen Peroxide method

 

MTT assay:

MTT Assay is a colorimetric assay⁴² that measures the reduction of yellow 3-(4, 5-dimethythiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase. The assay depends both on the number of cells present and on the assumption, that dead cells or their products do not reduce tetrazolium. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, dark purple coloured formazan crystals. The cells are then solubilized with a DMSO and the released, solubilized formazan reagent is measured spectrophotometrically at 570 nm.

 

 

DMEM (Dulbecco's modified Eagles medium), MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide], trypsin, EDTA Phosphate Buffered Saline (PBS) and were purchased from Sigma Chemicals Co. (St. Louis, MO) and Fetal Bovine Serum (FBS) were purchased from Gibco. 25 cm² and 75 cm² flask and 96 well plated purchased from eppendorf India. The cell lines purchased from NCCS (National Centre for Cell Science, Pune) and maintained in MEM supplemented with 10% FBS and the antibiotics penicillin/streptomycin (0.5mL^-1), in atmosphere of 5% CO₂ /95% air at 37⁰C. For MTT assay, each test compounds were weighed separately and dissolved in DMSO media make up the final concentration to 1 mg/ ml and the cells were treated with series of concentrations from 10 to 100µg/ml. Cell viability was evaluated by the MTT assay with three independent experiments with six concentrations of compounds in triplicates. Cells were trypsinized and perform the trypan blue assay to know viable cells in cell suspension. Cells were counted by hemocytometer and seeded at density of 5.0 X 10³ cells / well in 100 μl media in 96 well plate culture medium and incubated overnight at 37^oC. After incubation, take off the old media and add fresh media 100 µl with different concentrations of test compound in represented wells in 96 plates. After 48 hrs, discard the drug solution and add the fresh media with MTT solution (0.5mg/ml^-1) to each well and plates were incubated at 37 C for 3 hrs. At the end of incubation time, precipitates are formed as a result of the reduction of the MTT salt to chromophore formazan crystals by the cells with metabolically active mitochondria. The optical density of solubilized crystals in DMSO was measured at 570nm on a microplate reader. The percentage growth inhibition was calculated using the following formula.

 

The percentage growth inhibition was calculated by using the following formula:

 

                                                 Mean OD of Individual Test Group

% Growth of inhibition =    100 - -------------------------------------- x 100

                                                         Mean OD of Control Group

 

The IC₅₀ value was determined by using linear regression equation i.e. Y =mx+C.  Here, Y = 50, M and C values were derived from the viability graph.

 

Table 4: Cytotoxic activity (IC₅₀) of Synthesized compounds

S. No	Compound	IC5O (µg/ml)
		MCF-7	HepG-2	Hela
1	5e	59.65	49.57	53.87
2	9b	74.67	72.36	113.42
3	9g	49.58	48.36	28.58
4	13b	41.65	56.82	53.98
5	13c	12.79	38.32	56.25
6	13e	38.56	37.59	34.72
7	14b	72.34	85.37	48.37
8	14d	85.92	104.32	27.56
9	Imatinib	4.08	18.25	17.67

The pyridopyrimidine and pyrazolopyrimidine derivatives were designed, screened by molecular docking studies (Table 1) reveals that compounds may act as potent inhibitors of Tyrosine Kinase and Thymidylate synthase in comparison with imatinib and dasatinib (Figure 1 and 3) as standards. The compounds were optimized, synthesized (Figure 2) and characterized by spectral methods. Further experimental investigations such as antioxidant activity by DPPH method (Table 2) and Hydrogen peroxide methods (Table 3). Investigation of cytotoxic activity of the target compounds by MTT assay (Table 4 and Figure 4) was found to effective as reference imatinib.

 

 

Figure 4: Cytotoxic activity by MTT Assay

 

CONCLUSION:

According to the obtained data from docking studies compounds may act as potent targets of tyrosine kinase and thymidine synthases in comparison with standard imatinib. Further studies, antioxidant activity reveal that compounds 9g, 13c, 13e, 14b and 14d were potential compounds and MTT assay exhibited compounds 13c, 13e and 14d can be potential anticancer candidates against MCF-7, Hep G2 and Hela cell lines respectively. Based on all the studies conclude that good agreement was observed between the top-ranked docking scores and top experimental inhibitors. Hence, the compounds could be considered as new anticancer hits for further lead optimization.

 

ACKNOWLEDGEMENT:

The authors are thankful to Vels Institute of Science, Technology and Advanced Studies (VISTAS) and its management for providing research facilities and encouragement.

 

CONFLICT OF INTERESTS:

The authors declare that they have no conflict of interests regarding the publication of this paper.

 

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Received on 10.10.2019           Modified on 09.02.2021

Accepted on 23.04.2021         © RJPT All right reserved