Synthesis of Novel Fluorine Substituted Isolated and Fused Heterobicyclic Nitrogen Systems Bearing 6-(2’-Phosphorylanilido)-1,2,4-Triazin-5-One Moiety as Potential Inhibitor towards HIV-1 Activity

Novel 6-(5’-fluoro-2’-diphenylphosphorylanilido)-3-hydrazino-1,2,4-trizin-5 (2H) one (3) is achieved from hydrozinolysis of the corresponding 3-thioxo-analoges 2. Compound 2 is also obtained from phosphorylation of 6-(5’-fluoro-2’-aminophenyl)-3-thioxo-1,2,4-triazin-5(2H) one (1). Novel fluorine substituted isolated and/or fused heterobicyclic nitrogen systems bearing and/or containing, 6-phosphoryl anilido-1,2,4-trizin-5 (2H) one moiety (4 22) have been synthesized from ring closure reactions of compound 3 with π-acceptors activated carbon compounds in different medium and conditions. Structures of the products are characterized by MS, IR, UV-VIS, CH, N, and 1H/13CNMR spectral data. The new products have been evaluated as potential inhibitors towards HIV-1 activity.


Introduction
Organophosphorus systems are ubiquitous in nature and exhibit many applications in the field of agriculture medicine and industry [1] [2].Many multi-ring phosphorus heterocycles are used as pesticide [3], bactericide [4]- [6], antibiotics [4], and acts as HIV protease inhibitors [7].Thus, synthesis of new phosphorus bearing a heterocycles has attracted the attention of researchers.Phosphorylation of organic compounds often improves their biological activity, especially through a vital energy, because the P-O is the store of energy for metabolism process.For example, phophorylated-N in the nucleocapsid affects the interaction between the N-atom and the genomic RNA.The charge repulsion between the negatively charged phosphoserine and the negatively charged RNA may weaken the interaction between N-atom and RNA, thus enabling the viral polymerase to gain access to genomic RNA and to initiate viral RNA transcription and replication [8].On the other hand, chemistry of N-phosphorylheterocycles showed that these compounds from two dimensional polymeric chains via intermolecular P-O −+ H-N hydrogen bonds [9].Also, phosphodiester compound had a type of action, especially enzymetic of DNA replication [10] on DNA ligase as (Figure 1).
It is interesting that fluorine containing aheterocycles bearing functional groups exhibits highly effective in biological process, pharmaceuticals, agrochemicals, polymers and a wide range of consumer products [11].It reflects its resistance to metabolic change due to the strength of the C-F bond providing biological stability and the application of its nonstick-interfacial physical characteristics [12]- [16].Abdel-Rahman et al. [17]- [21] reported the synthesis and chemical reactivity of 3-thioxo-1,2,4-triazin-5 one derivatives as a bioactive molecules, especially as anti-cancer, anti-AIDS, Amyllolytic, cellobiase and antimicrobial agents.Based upon these observations, the aim of this work is to study the formation of 6-(5'-fluoro-2'-phosphoryl anilido-3-hydrazino-1,2,4triazin-5 (2H) one then study their behaviour as electron donor towards different electron acceptors such as carbo-sulfur, oxygen, halogen and nitrogen compounds; finally, a type of isolated and/or fused heterobicyclic systems obtained and evaluation as potential inhibitors towards HIV-1 virus.

Experimental
Melting points were determined with an electro-thermal Bibbly Stuart Scientific Melting point SMPI (UK).A perkin Elmer (Lambda EZ-2101) double beam spectrophotometer (190 -1100 nm) used for recording the electronic spectra.A Perkin Elmer model RXI-FT-IR 55,529 cm −1 used for recording the IR spectra (EtOH as solvents).A Brucker advance DPX 400 MHz using TMS as an internal standard for recording the 1 H/ 13 C NMR spectra in deuterated DMSO (δ in pp m).AGC-MS-QP 1000 Ex model is used for recording the mass spectra.Hexafluorobenzene was used as external standard for 19 FNMR at 84. 25 MHz and 31 P (in CDCl 3 , 101. 25 MHZ).
Elemental analysis was performed on Micro analytical Center of National Reaches Center-Dokki, Cairo, Egypt.Compound 1 prepared according the reported method [17].

Results and Discussion
α-Aminophosphonic acids continue to elicit study due to interest in their biological properties as herbicides [22], plant growth regulators [23] and most notably those species heaving a direct P-N bond are investigated as transition state analogues of the tetrahedral transition-state involved in peptide hydropysis [24].Ali et al. [25] [26] studied the reactivity of α-amino phosphonates as dipolar ion structure and have type of tautomeric formula due to the higher e-withdrawing of two phenoxy and P=O groups (Figure 2).Thus, α-aminophosphonate group had a higher degree of stability towards any attack of reagents, which attribute to presence of differ factors of stability [27].Phosphorus elements in these systems, was determine by using the spectrophotomeric.The method is based on the development of floated complex of molybdophosphonic acid (MPA) and methylene blue (MB) with N, N'-diphenylbenzamide (DPBA) in toluene and its subsequent dissolution in acetone [28].
Formation of 8 may be take place via aroylation then cycloadditon reaction [32] (Figure 4).The greater reactivity of the polyfunctional compound as aroylisothiocynate towards the hydrazino group as bi-nucleophile is presumably due to its favourable location between both carbonyl and thiocarbonyl functional   A large degree of the biological activity is attributed of the nature of substituent's and a degree of electronic distribution over the active center of the 1,2,4-triazines [34]  In view of interesting results obtained from the reaction of 1,3-oxazolium salts and of 1,3-oxazol-2-one derivatives with hydrazine derivatives [36], [37], it was worthwhile to investigate the behavior of oxazolone19 towards hydrazine-derivative. Similarly, 3-hydrazino-6-aryl-1,2,4-triazin-5 (2H) one ( 3  and molecular configuration as possible.In general, all the new compounds record the presence of n-π * , n-σ * , π-π * and σ-σ * electronic transition.UV-absorption spectrum of compound 3 as state recorded λ max (EtOH) at 346.48 nm, while that of compounds 14 (361.55)and 12 (361.12).A higher value of λ max for 14 and 12 than 3 is may be a lack's of -OH groups (which generate of H-bending (Figure 5).

Elucidation the Former Structures
On the other hand, UV absorption spectra of selected compound record a lower λ max than compound 3. λ max of 10 (330.56) and 18 (317.22)nm.A lower λ max of these compounds than the start is may be due to the presence of O-H group, which generate a type of H-bonding.The Keto-enol forms of 10 and 18 led to the inhibition of heteroconjugative, in addition a type of H-bonding which is closed to 1,2,4-triazine moiety (Figure 6).

IR-Absorption Study
The IR absorption spectral data show that most of the new compounds lack's a band of NH-P=O, which is due to a type of H-bonding present (Figure 6), while that of these compounds record only NH bond of new 1,2, 4-triazinones and/or pyrazoles moiety.In addition, IR absorption spectra of all the synthesized compounds exhibit an absorption bands at 3300 -3190 (NH), 1690 -1650 (C=O).Moreover presence of a characteristic bands at ν 1390 -1370 for cyclic NCN, 1250 and 1220 -1200 cm −1 for C-F and P=O functional.Also, all the new compounds record ν at 1100 -1050 cm −1 attribute to Ph-O-P group.Only, the compounds 3, 4, 6, and 7 record a ν for NH 2 at 3390 -3400 cm −1 , while that of the compounds 4, 6, 8, 10, 13, 16-21 showed ν for C=O in addition the original C=O of 1,2,4-trinzine.Finally, IR spectra of the compounds 4, 5, 6, 8-13 and 15, 16 record a type of bands characteristic for aliphatic groups (deformation 1480 -1440 cm −1 ).

NMR Spectral Study 1) 1 H NMR Spectral Study
The NH proton signals in all the new compounds appear as doublets at δ 8.8 -8.2 ppm (JP-N-H=6 -5.5 H z ) is due to its coupling with phosphorus.Also, H-bonding with oxygen of P=O and the deshielding effect of phenoxyphosphoryl group (Ph-O-P=O) are obviously the contributing factors for the downfield shift of the NH proton.On the other hand, phenoxy protons resonated at δ 7.50 -7.1 ppm and their integration corresponds to five protons with no splitting of the signals.This shows that all the protons as magnetically equivalent.Normally, exo and endo NH protons of 1,2,4-triazinone reveal at δ12 -11 ppm, while what of the 1,2,4-tiazinone addujent of CH 2 or NH protons show as enolic protons at δ11 -10 ppm (5,7,17,18,9).In addition, all the new compounds 4 & 6, exhibited a resonated signals at δ5 -4 ppm as NH 2 protons and NH proton at δ11 -10 ppm.Finally, the perhydro pyrazolyl-1,2,4-triazinones and the 1,2,4-trinzino-1,2,4-trinzinones which containing an aliphatic protons show a resonated signals at δ4 -3 and 1 -0.5 ppm for CH 2 and CH 3 protons.
2) 13 CNMR Spectral Study The 13 C Chemical shifts of phenoxy moiety are agreeing well with the reported values [38].But, the coupling constants for 2 Jare concurring with those of equationally oriented P-O-Ar groups [39] showing the 1,2,4-trinzine ring has probably half chair conformation with phosphorus atom projecting upwards and the O-Ar group orienting equationally.The carbons C, which are connected to phosphorous through NH, resonated at δ 112.97 with 2 J P-N-C (d, J 8.1) and the difference in their chemical shifts may be attributed to the variation of shielding effect of NH [40].In addition, all the new compounds record the resonated attribute to C=O (170 -160), C-F (150 -140), C=N(140 -130), C-N (111 -110) of 1,2,4-triazinone, with a differ type of carbons of pyrazolyl as well as carbons of other 1,2,4-triazine formed (Figure 7 and Figure 8).

Mass Spectrometric Measurements
The mass spectral investigations of the isolated heterobicyclic system is differ than the fused heterobicyclic systems (14 & 18) for example, M/Z of 7 and/or 10 recorded the molecular ion Peak's at m/z 534 and 336 respectively with a lower abounds percent's, which indicating the fragile nature of these systems.While M/Z of compound 14 showed the highest value peak at 579 with a base peak at m/z 95, which give us a high degree of stability for this Skelton.Moreover, M/Z of 18 exhibits a molecular ion peak at m/z 522 with moderate abounds percent.From these data, we can conclude that fused heterobicyclic nitrogen systems are more stabilized [41] Figure 7. 13 C NMR data of compound 10.than other isolated heterobicyclic systems.It's interest that in all mass fragmentation pattern, 4-fluorophenyl ion is a base peak followed by N-phosphorus oxide ions, while that heterocycle supported that a large fragmentation bath way (Figures 9-12).Figure 8. 13 C NMR data of compound 18.

HIV-1 Inhibition (Enzyme Inhibition)
Human immunodeficiency virus type-1 is the causative agent of acquired immunodeficiency syndrome (AIDS) which is one of the most serious health problems [42].Since reverse transcriptase (RT) is an essential enzyme for the replication [43] of HIV, it is the most favoured target for the antiviral chemotherapy against HIV infection [44].3'-Azido-2',3'-dideoxythymidine (AZT) [45] and 2',3'-dideoxyinosine (DDI) [46], 2',3'-dideoxycytydine (DDC) [47] and 2',3'-didehydro-3-deoxythymidine (DT4) [48] are the well-known potent nucleoside reverse transcriptase inhibitors clinical use, but unfortunately they produce serious side effects such as bone marrow suppression.The search for a more effective and less toxic agent has brought into focus potent yet structurally different non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) [49].Shakil et al., [50] reported that increase or decrease of electro-negativity and hydrophobicity of the bioactive drugs, cytotoxicity will also increase or decrease accordingly.So less electronegative and less hydrophobic substituents would be preferred to design the less cytotoxic drugs.The large number of research papers published every year indicate that the development of an effective drug for the inhibition of HIV-1 via enzymes inhibitors [51] [52].HIV PIs for example, prevent the cleavage of the gag and gag-pol precursor polyproteins to the structural proteins and functional proteins, thus arresting maturation and thereby blocking infectivity of the nascent virions.e.g.Tipranavir showed loss cross-resistance to HIV strains that were resistant to the established (peptidomimetic) inhibitors of HIV protease.Also, tipranavir retained marked activity against HIV-1 isolates derived from patients with multidrug resistance to other PIs (Figure 13).In search for new poly substituted 1,2,4-triazine bearing a phosphoryl group.The present work is synthesize novel fluorine substituted phosphorylanilido-1,2,4-triazin-ones and evaluate as potential inhibitors for HIV-1.The procedure used in the National Cancer Institute's Test for agents active against HIV is designed to detect agents acting at any stage of the virus reproductive cycle [53].The assay basically involves the killing of T 4 lymphocytes by HIV.Small amounts of HIV are added to cells and two cycles of virus reproduction are necessary to obtain the required cell killing.Agents that interact with virions, cells or virus gene-products to interfere with viral activities will protect cells from cytolysis.The tetrazolium salt XTT is added to all wells and cultures are incubated to allow formazan color development by viable cells used analyzed spectrophotometrically (Figure 14).

Figure 1 .
Figure 1.The reaction of DNA ligase phospho diester link.

Figure 2 .
Figure 2. A possible present formula of the new synthesized isolated systems.

Figure 5 .
Figure 5. UV-absorbtion data of compound 3 and some prepared compounds.

Table 2 .
The in vitro anti-HIV-1 screening results of compound 18.