Fluorine Substituted 1 , 2 , 4-Triazinones as Potential Anti-HIV-1 and CDK 2 Inhibitors

Fluorine substituted 1,2,4-triazinones have been synthesized via alkylation, amination, and/or oxidation of 6-(2-amino5-fluorophenyl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one 1 and 4-fluoro-N-(4-fluoro-2-(5-oxo-3-thioxo-2,3,4,5-tetrahydro1,2,4-triazin-6-yl)phenyl)benzamide 5 as possible anti-HIV-1 and CDK2 inhibitors. Alkylation on positions 2 and 4 in 1,2,4triazinone gave compounds 6–8. Further modification was performed by selective alkylation and amination on position 3 to form compounds 9–15. However oxidation of 5 yielded compounds 16–18. Structures of the target compounds have been established by spectral analysis data. Five compounds (5, 11, 14, 16, and 17) have shown very good anti-HIV activity in MT-4 cells. Similarly, five compounds (1, 3, and 14–16) have exhibited very significant CDK2 inhibition activity. Compounds 14 and 16 were found to have dual anti-HIV and anticancer activities.


Introduction
Human immunodeficiency virus (HIV) type-1 is the causative agent of acquired immunodeficiency syndrome (AIDS), which is one of the serious global health problems [1].The currently approved anti-HIV drugs can be divided into five groups: reverse transcriptase inhibitors (RTIs), protease inhibitors [2] (PIs), fusion inhibitors (FIs), coreceptor inhibitors (CRIs), and integrase inhibitors (INIs).This arsenal of drugs, which is used in combinations, has moved the prognosis of HIV patients from that of high morbidity and mortality to, for many at least, a chronic, manageable but still complex disease [3][4][5].However, the use of these drugs has been relatively limited by their toxicity [6], drug resistance development [7], and, more worryingly, the fact that some newly HIV-infected patients carry viruses that are already resistant to the currently approved AIDS treatments [8].These issues along with drug-related side effects make it apparent that new anti-HIV drugs with novel mechanisms of action are clearly needed.
On the other hand, during the past 30 years, a variety of approaches have been taken for cancer chemotherapy and many antitumor drugs have been developed for clinical use.In the treatment of solid tumors, however, the conventional approaches have met with only limited success and cancer still remains as one of the leading causes of human mortality [9].
Chemotherapy drugs are sometimes feared because of a patients' concern about its toxic effects.There are three goals associated with the use of the most commonly used anticancer agents: (a) damage the DNA of the affected cancer cells, (b) inhibit the synthesis of new DNA strands to stop the cells from replicating, and (c) stop the mitosis or the actual splitting of the original cell into two new daughter cell [10].
In the past few years fluorine substituted heterocyclic nitrogen systems have been incorporated into drug discovery research [11][12][13][14][15][16][17][18][19][20][21][22][23] to improve the physicochemical properties of drugs.Organic fluorine is a prominent tool in the design and improvement of pharmacokinetic properties of drug molecules.Replacing hydrogen and other functional groups with fluorine can have a dramatic effect on the modulation of electronic, lipophilic, and steric parameters, all of which can critically influence both the pharmacodynamic and pharmacokinetic properties of drugs.Substitution of fluorine into a potential drug molecule not only alters the electronic environment, but it also influences the properties of neighboring functional groups.Fluorine can have significant effects on the binding affinity in protein-ligand complexes.The DNA polymerase inhibitors fludarabine (I) (F-ara-A), clofarabine (II), and tezacitabine (III) are used as cancer chemotherapeutic agents [24], while Gleevec (IV) is used as catalytic inhibitor of imatinib mesylate [25].In addition, BX-1382BS (V) showed a significant activity as protein kinase inhibitor [26] and a cyanopyrimidine scaffold JNJ-17029259 (VI) is an oral inhibitor of VEGF-mediated signal transduction [27] (Figure 1).Recently a great deal of synthetic efforts has been spent on fluorinated uncondensed 1,2,4-triazines by our group searching for new anti-HIV and anticancer agents [28][29][30][31][32][33][34][35][36][37][38] (compounds A-F; Figure 2).
Fluorine incorporation on key positions plays a significant role to alter the physicochemical and biological characteristics of organic compounds.Frequently, it is found that a fluorine substituent leads to an enhancement of the binding affinity of a molecule with proteins through a noncovalent bond formation.Fluorine increases binding affinity, reduces plasma protein binding leading to a higher free fraction of the drug, and increases cell penetration.The combination of these effects results in a dramatically improved biological activity.Based on these valuable observations and in part of our continuing efforts in drug development, the present work describes an attempt towards the synthesis of fluorine substituted 1,2,4-triazinones.The purpose of the work is to extend the scope of our previous studies [29,30] and to substitute fluorine on various positions in 1,2,4-triazines in order to obtain effective anti HIV-1 and CDK2 inhibiting agents.

Experimental
2.1.Chemistry.Melting points were determined on an electrothermal Bibby Stuart Scientific melting point apparatus and are uncorrected.The infrared (IR) spectra were recorded on PerkinElmer RXI FT-IR infrared spectrophotometer using the KBr pellet technique.Electronic absorption spectra were recorded in DMF on Shimadzu UV-Visible 3101 PC spectrophotometer. 1 H and 13 C NMR spectra were recorded on a Bruker DPX-400 FT NMR spectrometer using tetramethylsilane as the internal standard DMSO- 6 as a solvent (chemical shifts in , ppm). 19F NMR spectra were determined at 84.25 MHz using hexafluorobenzene as an internal standard.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 PerkinElmer Series 2 analyzer.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 at  254.

Anti-HIV-1 Activity.
All the new synthesized compounds have been evaluated for their in vitro anti-HIV activity that was performed on T-4 lymphocytes infected and uninfected with HIV-1 using DMSO as solvent.The assay involves the killing of T-4 lymphocytes by HIV.Uninfected cells with the compound serve as a toxicity control, and infected and uninfected cells without the compound serve as basic controls.Cultures are incubated at 37 ∘ C in a 5% carbon dioxide atmosphere for 6 days.The tetrazolium salt, XTT, is added to all wells, and cultures are incubated to allow formazan color development by viable cells.Compounds that degenerate or are rapidly metabolized in the culture conditions may not show activity in this screen.Zidovudine (AZT) at 10 M was used as a control.The viability of the cells was determined spectrophotometrically to quantitate formazan production and in addition is viewed microscopically for detection of viable cells and confirmation of protective activity.Drug-treated virus-infected cells are compared with drug treated noninfected cells and with other appropriate controls (untreated infected and untreated noninfected cells, drugcontaining wells without cells) on the same plate.

CDK2 Inhibition
Assay.CDK2-cyclin E kinase was expressed and assayed as previously described [39].Kinase activity was expressed as a percentage of maximum activity.The concentration of the test compounds required to decrease the CDK activity by 50% was determined from dose-response curves and designated IC 50 .The IR of 1 showed strong peaks at 3528 and 3300 cm −1 for NH, NH 2 and at 1661 and 1255 cm −1 for C=O and C-F functional groups, respectively.The mass spectrum showed a peak at m/z 254 (M + H 2 O, 5%) with a base peak at 103.Similarly, compound 3 exhibited in its IR spectrum three peaks at 3310, 2929, and 2862 cm −1 for NH and methyl groups.The mass of 3 exhibited a peak at m/z 284 (M + H 2 O, 5%) with a base peak at 88.The 1 H NMR spectra of 3 showed two singlets at  3.2 and 3.5 ppm for N-CH 3 and S-CH 3 protons.
Reaction of 1 with 4-fluorobenzoyl chloride in dry pyridine furnished 5 (Scheme 3).IR spectrum of 5 showed two carbonyl groups at 1663 and 1610 cm −1 .The 13 C NMR spectrum showed peaks at  173.15, 164.14, and 158.72 ppm attributed to C=S, C=O, and CONH carbons.The mass spectrum exhibited a peak at m/z 360 (M + 4, 6%) with a base peak at 89. Compound 5 was used as starting material for building some more N 2 -substituted and/or C 3 -NH substituted 1,2,4triazinones.Thus hydroxymethylation of 5 using methanolformaldehyde yielded 2,4-dihydroxymethyl derivative 6 which on its treatment with secondary amine as morpholine and/or 2,2-dipyridylamine under the same reaction conditions Mannich bases of the type 7 and/or 8 were isolated (Scheme 3).IR spectrum of 6 showed absorptions at 3537, 2935, 2890, 1498, and 1440 cm −1 mainly attributed to O-H, C-H stretching, and CH 2 bending vibrations.The 1 H NMR spectrum of 6 showed peaks at  12.54, 10.15 and 8.04 ppm for NH and OH protons.The 13 C NMR spectrum, however, exhibited three resonance signals at  164.99, 163.32, and 157 ppm for C=S, C=O, and CONH groups and at  39.0 ppm for CH 2 carbon.
Structure of 13 was deduced from elemental analysis and spectral data.The IR spectrum showed peaks at 3550-3100, 2919, 2882, 1658, and 1610 cm −1 attributed to O-H, N-H, CH 2 , C=O, and CONH groups.In 1 H NMR spectrum signals at  14.45, 12.58, and 10.25 ppm for NH, NH, and OH protons.In addition to this, a peak at  3.96 appeared for CH 2 protons.Further support was given by its 13 C NMR where there are three peaks for three carbonyl groups at  168.74, 164.99, and 159.27.The mass spectra recorded a peak at m/z 418 (M-28, 390, 1.00%) with a base peak at 149 as C 8 H 4 NOF.The IR spectra showed absorptions at 3534, 3300, 1661, and 1640 cm −1 for NH 2 , NH, and C=O functional groups.The spectrum did show two peaks at 1255 and 658 cm −1 for C-F group.The 13 C NMR spectrum lacks a signal for C=S carbon.
Oxidation of compound 5 by refluxing with sulfur flowers in dry benzene yielded the disulfide 16 (Scheme 5).Due to higher nucleophilicity of sulfur when compared with oxygen and nitrogen, the removal of H + from S-H is easily followed by removal of H − from other molecules to form a disulfide 16 with evolution of hydrogen (Scheme 5).The IR spectrum showed an absorption band at 1090 cm −1 for C-S-S-C group.The mass recorded m/z 341 (M-18, -H 2 O, 85%) with a base peak at m/z 306 for C 16 H 5 N 3 SOF.Finally synthesis of fluorine substituted 1,2,4-triazinone bearing other amino-1,2,4-triazinone 18 was done by refluxing compounds 1 and 15 in boiling isopropyl alcohol as amination reaction (Scheme 5).The IR spectrum of 18 showed bands at 3320, 3300, 1668, 1620, and 1185 cm −1 for NH, NH, C=O, CONH, and C=S groups.The mass spectrum showed a peak at m/z 368 (M-196, 7%) along with a base peak at 89 for CH 2 N 3 S.

Journal of Chemistry
The presence of fluorine was confirmed by 19 F NMR.A single fluorine atom attached to phenyl and/or benzoyl ring appeared in the region  120-130 ppm using hexafluorobenzene as internal standard.
The mass fragmentation patterns of some of the target molecules are mentioned in Schemes 6, 7, 8, 9, and 10. infected with HIV using DMSO as solvent.The assay basically involves the killing of T4 lymphocytes by HIV compounds that degenerate or are rapidly metabolized in the culture conditions may not show activity in this screen.The viability of the cells was determined spectrophotometrically using the tetrazolium assay procedure.The results obtained are reported in Table 1.

Biological Evaluation
The results revealed by Table 1 suggest that compounds 5, 11, 14, 16, and 17 displayed very good anti-HIV-1 activity having a favorable selectivity index between 3 and 7.The CC 50 and EC 50 data are used to calculate the selectivity index (SI) of each compound as an estimate of a therapeutic window and a mechanism to identify candidates for efficacy studies.The other compounds though are active as anti-HIV-1 agents but do not show very good selectivity index ratio.The remaining compounds exhibited an average to poor activity with selective index <3 (Table 1).In particular, a high activity level was observed for compounds 11 and 14.A close examination of these two compounds reveals that both the structures have 4-fluoro-N-(4-fluorophenyl)benzamide part connected at position 6 in triazinone nucleus.Similarly, in compound 16 which is an oxidised dimer of the active compound 5, the selectivity index is almost doubled.The antiviral activity diminishes if the triazinone ring experience crowding at carbon-3 in triazinone part as in case of 15 and 18. Sulfonamide moieties, however, could not contribute towards improving the antiviral profile; rather it significantly diminishes the selectivity index ratio as can be seen in compounds 9 and 10.
The influence of fluorine on the acidity, hydrogen bonding, and lipophilicity of these systems can be envisaged not only a biological activity modulator but also influences the bioavailability of the drug.The active fluorine compounds obtained are capable of forming stronger DNA complexes than their nonfluorinated analogs.These results suggest that the electronic nature of the chain of 3-thioxo-1,2,4-triazines tethering an intercalator that not only influences the DNAbinding process but might also be used to tune the new DNA-drug complex.It can be implied that the fluorine substituted-3-thioxo-1,2,4-triazin-5-ones represent suitable prodrug principle leading to higher bioavailability.
The biological activity depends not only on the site of fluorination and the geometry of the conjugate carbanion formed but also on the total electronegativity of new heterocyclic nitrogen systems.In view of these characteristics and the results obtained in Table 1 we can infer that fluorination invariably increases C-H acidity through a combination of inductive and hyperconjugative resonance stabilization of the carbanion and thus influences the biological activity of this series of compounds (Figure 3).
The results from Table 2 reveal that compounds 1, 3, and 14-16 show very significant CDK2 inhibitory activity.Compound 3 was found to be as active as olomoucine, while compounds 1 and 14-16 were more potent than the standard compound.Once again the same structural features are present in active compounds as is evident in compounds active as anti-HIV-1.

Conclusions
According to the data obtained from the biological assay five compounds 5, 11, 14, 16, and 17 have shown very good anti-HIV activity in MT-2 cells.Similarly, five compounds (1, 3, and 14-16) have exhibited remarkable CDK2 inhibition activity.Compounds 14 and 16 can be considered as a significant matrix for the design and synthesis of novel candidates with dual anti-HIV and anticancer activities.Further investigation into the other aspects of structure activity relationship studies of this series of compounds is required in order to explore the scope and limitation of its biological activities.