Synthesis of Barbituric and Thiobarbituric Acids Bearing 5,6-Diphenyl-1,2,4-Triazin-3-yl Moiety as CDK2 Inhibitors of Tumor Cells

Synthesis of several new diphenyl-1',2',4'-triazin-3'-yl barbituric acid are described. The method involves addition reaction of isocyanate and isothiocyanate and 3-amino-5,6-diphenyl-1,2,4-triazine (1) to give N,N-disubstituted urea 2 and N,N-disubstituted thioureas 3 and 4 respectively. Further, ring closure reactions with malonate ester give barbituric acid 5 and thiobarbituric acid 6 and 7. The Presence of the active methylene in the skeleton of compound 5-7 at C-5 are deduced by condensation with pyridine-4-carboxyladehyde to give barbituric and thiobarbituric acids (8-10). Further fluoroacylation of compounds 5-7, afforded 1-(cyclohexyl/methyl/phenyl)-3-(5',6'-diphenyl-1',2',4'-triazin-3'-yl)-5-(trifluoracetyl)-5Hbarbituric/thiobarbituric acids (11-13). Synthesis compounds of the series 5-(trifluoroacetyl) barbituric acid (11) and 5(trifluoroacetyl) thiobarbituric acids (12 and 13) were able to inhibit activity of CDK2 in a biochemical assay with IC50 values comparable to olomoucine. In addition, a pyridine side chain at C-5 (compound 9 and 10) significantly decreases CDK2 inhibitory activity.


Introduction
The success of Imatinib mesylate as the first small molecule targeted kinase inhibitor for use in cancer therapy [1], are validated protein kinases as important drug targets in the treatment of human diseases [2]. The protein kinase family constitutes the largest gene-family for therapeutic development, and hence there is an urgent need to develop and discover compounds that can both serve as pharmacological probes and lead compounds for further drug development [3]. The condensed and substituted thiobarbituric acids possesses diverse pharmacological profile such as antimicrobial, selective cell adhesion inhibitors and DNA cleavage activities [4]. Additionally Barbiturate and thiobarbiturate derivatives attracted considerable attention owing to their various biological effects such as inhibiting collagenase-3 (MMP-3), recombinant cytochrome P450 enzymes and antiinflammatory analgesic [5]. In focused the Polyfunctional heterocyclic nitrogen systems are essential for drug discovery [6], For example, pyrazolyl-1,2,4-triazines [5], 3functionalized-5,6-diphenyl-1,2,4-triazines [6] and 3-amino-1,2,4-triazines [7]. In addition, functionalized bridgehead nitrogen heteroannulated 1,2,4-triazine systems works as pharmacological probes [10] while the pyrimidine nucleus and their derivatives exhibit a wide range of pharmacological, medicinal and biological properties [11].

Results and Discussion
This work reports a simple synthesis strategy toward both 5,6-diphenyl-3-amino-1,2,4-triazine (1) and the substituted barbituric/thiobarbituric acids, Structures of the new targets obtained established from their correct elemental analysis and spectral measurements.
According to the condensation of compound 6 with pyridin-4-carboxaldehyde the active methyl group (C-5) not showed as expected in the IR, 1 H NMR and 13 C NMR spectra of compounds 8-10, ppm. Furthermore, IR spectrum showed only two carbonyl groups at ν 1720, 1700 cm -1 and (C=S) at On the other hand, fluoroacetylation of compound 6 by reflux with hexafluoroacetic anhydride in THF afforded the mono (trifluoroacetyl) derivative 12. Structure of compound 12 confirmed by rang of spectrum analysis, IR spectrum showed broad hydroxyl peak of (OH) at 3450-3400, carboxylic C=O at 1700 cm -1 , while aliphatic CH at C-5 appears at 1414 cm -1 . thiobarbituric acid (C=S) at position-3 showed peaks at 1180 cm -1 . 13 C NMR of 12 recorded the presence of resonated carbon signals at δ 180, 166, 146, 142, 138, 132-122 and 40 ppm for C=S, C=O, C-F, C=N, C=C, CH 3 respectively, in addition to aromatic carbons of 1,2,4triazine moiety.
Mass fragmentation of compound 12 showed m/e at lower percentage, which may be a less stability of the fluorinated systems, with a base peak at m/e 85 attribute to COCF 3 ions.

Experimental
Melting points were determined with an electrochemical Bibly Sturat Scientific melting point sample (UK). A Perkin Elmer Model RXI-FT IR system 55529 was used for Recording IR spectra of the prepared compounds (cm -1 ). the 1 H and 13 CNMR spectra DMSO-d 6 (ppm), A Bruker advance DPX 400 MHZ model uses TMS an internal standard. A GC-MS-GP 1000 Ex model was used for recording the mass spectra of the compounds (MHz). Electronic spectra were recorded in ethanol on Shimadzu UV and visible 310 IPC Spectrophotometer (nm). Elemental analysis was performed in micro analytical Center of Cairo University, Cairo, Egypt. 3-Amino-5,6-diphenyl-1,2,4-triazine (1) prepared according the reported method [7].

Inhibition of Cyclin-Dependent Kinase 2 (CDK2) for Cell Tumor Division
The purine-related pyrrolo[2,1-f] [1,2,4]triazines have been identified as tyrosine kinase inhibitors, a well-established platform for modern anticancer chemotherapy [15]. Recent control on the cell tumor division depends on use of polyfunctional heterocyclic nitrogen systems as CDK2 for examples fluorine substituted thiobarbituric acid [16].
Generally, this investigation showed that Thiobarbituric acids 6 and 7 showed activity over than barbituric acid 5 and their thiourea derivatives 3 and 4. While trifluoroacetylthiobarbituric acid 12 Record higher activity over the corresponding thiobarbituric acid 6. Furthermore, compound 12 had a higher percentage of fluorine elements, which led to a higher hydrophobic, distribution, and H-bonding formation. Table 1.

Conclusion
Fluorinated thiobarbituric and barbituric acids and their related systems, have been synthesis by simple effective methodology by the addition of isocyanate and isothiocyanate to 3-amino-1,2,4-triazine followed by ring closure reactions with malonic acid and finally condensation with fluoroacetylation. The novel synthesized systems is preliminary evaluated as CDK2 for cell tumor division, compounds with sulfur and trifluoroacetyl exhibit a higher activity over standard and other synthesis compounds.