Synthesis of Fluorinated Heterobicyclic Nitrogen Systems Containing 1 , 2 , 4-Triazine Moiety as CDK 2 Inhibition Agents

New fluorine substituted heterobicyclic nitrogen system as imidozolopyrimidines (2,3), pyrimido1,2,4-triazinones (4-7), 1,2,4-triazinyl-1,2,4-triazine (12-16), 1,2,4-triazinyl-1,2,4-triazinones (1417) and substituted thiobarbituric acids (19-20), have been synthesized using the reaction of 3amino-5,6-di (4′-fluorophenyl)-1,2,4-triazine (1) with α,β–bifunctional compounds. Structures of the title compounds were characterized by UV, IR, 1H/13C-NMR and mass spectrometric method. The studied compounds were tested for CDK2 inhibiting activity in DNA damage, as well as in vitro anti-tumor activity.


Introduction
1,2,4-triazines and their condensed analogues have shown to display wide-ranging applications in medicinal and pharmaceutical chemistry.The NCNN group of 1,2,4-triazine ring is an essential part in various biological activities.Tirapazamine as antitumor [1], Lamotrigine as anti-epileptic drug [2], and fused 1,2,4-triazines as antimicrobial [3]- [5], anti HIV [6] antimycobacterial [7], antiviral [8] [9], anxiolytic [10] and antidepressant [11] agents are already reported in literature.Heterobicyclic nitrogen systems containing 1,2,4-triazine moiety have also shown anti-HIV and anticancer activities [12]- [16].However, introduction of fluorine atoms to these bioactive heterocycles often improves their pharmacological properties mainly due to increased membrane permeability, enhanced hydrophobic bonding and stability against metabolic transformation owing to the strength of the C-F bond [17]- [21].Also, introduction of fluorine in these heterobicyclic systems exhibited enhanced anti-tumor activity [20].The search for novel cancer treatment has however entered a new post-genomic era and the emphasis now is on influencing cell signaling mechanisms such as those triggered by kinases [22] where selective inhibition of the Kinome constituents and related signaling proteins has opened up a diversity of new drug targets.
Cyclin-Dependent Kinases (CDKs) are families of serine/threonine kinases that play a well-established role in the regulation of the eukaryotic cell division cycle and have also been implicated in the control of gene transcription and other processes [23].Dysregulation of cell cycle in cancer cells has provided a rationale for the development of small molecule inhibitors of CDK1 as novel anticancer drugs.It was believed that CDK2 was the master regulator of S phase entry.Gene knockout mouse studies of cell cycle regulators revealed that CDK2 is dispensable for S phase inhibition and progression whereby CDK1 can compensate for the loss of CDK2 and the latter was found to be involved in cell cycle independent functions such as DNA damage repair [24].
Rational approaches to the design of fluorine-containing potential inhibitors associated with the strategic placement of fluorine in these molecules led us to design CDK2 inhibitors as possible anti-cancer agents.The aim of the present work is to synthesize and develop fluorinated fused and/or isolated heterobicyclic nitrogen systems, derived from the interaction between 3-amino,6-(4′-fluorophenyl)-1,2,4-triazines (1) and α, β-bifunctional oxygen, sulfur, halogen and nitrogen compounds.In this work, we evaluate the protein kinase inhibiting and cytotoxic activity of fluorinated heterobicyclic systems containing 1,2,4-triazine moiety.

Chemicals and Methods
Melting points were determined using an electrothermal Bibby Stuart Scientific melting point apparatus and are uncorrected.The infrared (IR) spectra were recorded on a Perkin-Elmer FT-IR infrared spectrophotometer using the KBr pellet technique.Electronic absorption spectra were recorded in DMF using a Shimadzu UV-Visible 1650 PC spectrophotometer. 1 H and 13 C NMR spectra were recorded on a Bruker DPX-400 FT NMR spectrometer using tetramethylsilane ( internal standard DMSO-d 6 ) as a solvent (Chemical shifts in δ, ppm). 19F NMR Spectra were determined at 84.25 MHz using hexafluorobenzene as a solvent.Splitting patterns were designated as follows: s: singlet; m: multiplet.Mass spectra were measured on a GCMS-Q 1000 Ex spectrometer.Elemental analyses were performed on a 2400 Perkin Elmer Series 2 analyzer and the found values were within ±0.4% of the theoretical values.Follow up of the reactions and checking the homogeneity of the compounds were made by TLC on silica gel-protected aluminum sheets (Type 60 F254, Merck) and the spots were detected by exposure to UV-lamp.
Nucleophilic attack on exo C=N bonds of 1,2,4-trizaine provides a direct and convenient method for functionalization of 1,2,4-triazines, which makes it possible to introduce various substituents in a single reaction.Thus, it is quite probable that their reactions with bifunctional nucleophiles would result in polycyclic compounds via attack by the second nucleophilic center at another carbon atom of hetero-ring [26].
The presence of hetero atoms results in significant changes in the cyclic molecular structure, due to the availability of unshared pairs of electrons on nitrogen atoms and the difference in electronegativity between heteroatoms and carbons within the closed system.
X-ray analysis of 1,2,4-triazine ring reveal that the ring is slightly distorted due to the asymmetry induced by the electronegativity of nitrogen atoms and two intermolecular hydrogen bonds that stabilized the 1,2,4-triazine structure [28].Scheme 1. Synthesis of 2 and 3.
Structure of compound 2 showed a characteristic bands at 3350 and 1623 cm −1 attributed to NH 2 group while that of compound 4 had a characteristic band at 1660 cm −1 for C=O, in addition to bands at 2950 and 1480 cm −1 for CH 2 group and an additional absorption at 1226 cm −1 for C-F.Mass spectra of 3 showed a molecular ion peak at m/z 323, with a base peak at m/z 214 for 4,4‫-׳‬difluorophenyl acetylene radical [29].
Ring closure reactions of compound 1 with diethyl malonate and/or ethyl cyanoacetate in refluxing THF afforded 1,2,3,4-tetrahydro-7,8-di(4′-fluorophenyl)-pyrimido [3,2-b] Structures of both compounds 4 and 6 were elucidated from their analytical and spectral data.IR spectrum of 4 showed absorption bands at 1680 and 1650 cm −1 for the two C=O groups, while that of 6 recorded absorption bands at 3300, 1670 cm −1 due to NH 2 and C=O groups, respectively.Mass spectrum of 6 recorded a molecular ion peak at 351 m/z and a base peak at 214 m/z.UV absorption spectrum of 5 showed a higher maximum (λ max = 351 nm) than that of 4 (λ max = 346 nm), due to the extended conjugation in the heterocyclic ring in the former. 1 H-NMR of 5 showed a prominent peak at δ 8.2 ppm due to C=CHAr. 13C-NMR of compound 7 showed the resonance signal of exo N=CH in addition to an endo N=C of 1,2,4-triazine.
The treatment of compound 1 with ethyl chloroformate in benzene and triethylamine and/or CS 2 /KOH led to the formation of ethyl carboxylate 8 and the intermediate 9, respectively.Hydrazinolysis of 8 and 9 by refluxing with hydrazine hydrate in ethanol afforded N 4 -substituted semicarbazide/thiosemicarbazide 10 and 11, respectively (Scheme 3).IR spectra of both compounds 10 and 11 showed C=O and C=S at 3400, 3100 cm −1 for NH, NH 2 groups, respectively.
IR spectra of 12 and 14 evealed absorption bands at 3150 -3100 and 1680 -1660 cm −1 for NH and C=O groups, respectively. 1H-NMR spectrum of 14 showed resonance signals at δ 1.95, 13.48 and 8-6.6 ppm for CH 3 , NH and aromatic protons respectively.However compound 13 recorded a resonance C=S at δ 195 ppm.Mass spectra of 13 exhibit a molecular ion peak at m/z 384 and a base peak at m/z 214 due to 4,4'-difluorophenyl acetylene radical [29].
Under the same experimental conditions, ring closure reactions of compound 11 with triethylorthoformate, carbondisulfide and sodium pyruvate resulted in 3-(2′,3′-dihydro-3′-thioxo-1′,2′,4′-triazol-4′-yl)-5,6-di(4′-fluo-Scheme 2. Synthesis of 5 and 7. Fluorinated N,N-disubstituted-thiobarbituric acid 19 was prepared from refluxing compound 9 with 1 in ethanol to give N,N-disubstituted-thiourea 18. Ring closure reaction of 18 with malonic acid on warming with glacial acetic acid yielded compound 19 (Scheme 6).IR spectra of compound 18 showed absorption bands at 3300 -3190 cm −1 for NH, NH of thiourea derivative which was not found in compounds 19 and 20. 1 H-NMR spectrum of compound 19 recorded a resonance signal at δ 2.44 ppm for CH 2 protons.Also, 13 C-NMR showed a signal at δ 24.88 ppm for aliphatic carbons.IR spectrum of 20 showed absorption bands at 1680, 1660 and 1611 cm −1 for the two C=O and CH=C groups, in addition to two bands at 1255 and 1185 cm −1 for C-F and C=S groups, respectively. 13C-NMR of 20 exhibited resonance signals at δ 188, 166 and 39.48 ppm attributed to C=S, C=O and CH=C carbons, respectively.It is interesting to note that UV absorption of compound 20 recorded λ max at 334 nm which is higher than the value for compound 19 (λ max = 332), confirming ta presence of an α, β-unsaturated cyclic ketone system in the former.

Biological Evaluation
The CDK2 inhibitory activity of the synthesized compounds revealed that eleven out of the tested twenty compounds displayed variable inhibitory effects.However compounds 11, 13, 16 and 17 showed profound activity.

Conclusion
The synthesized α, β-bifunctional oxygen, sulfur, halogen and nitrogen derivatives of 1,2,4-triazines have shown promising CDK2 activities.Out of eleven active compounds, four of them (11, 13, 16 and 17) have shown very good CDK2 enzyme inhibiting activity.In these compounds N-(amino(hydrazinyl)methyl)hydrazinecarbothioamide scaffold was found to be an essential molecular feature in CDK2 enzyme inhibition activity.However these four compounds have also shown significant activity against various tumor cell-lines in subpanel assay.

Table 1 .
The CDK2 inhibitory activity of tested compounds (IC 50 in µg/ml).The data represent means values from three independent experiments plus the standard deviation (SD).

Table 2 .
In vitro antitumor activity data of some active compounds.
GI50: concentration giving 50% inhibition; TGI: concentration giving total growth inhibition; LC50: concentration having 50% lethal effect; Δ is considered low if 1, moderate if > 1 and high if ≥ 3; Subpanels showing a statistical measure of differential sensitivity with respect to the indicated response parameters.