Synthesis of Some New Fluorine Substituted Thiobarbituricacid Derivatives as Anti HIV 1 and Cyclin-Dependent Kinase 2 ( CDK 2 ) for Celltumer Division-Part II

In search for new potential inhibitors some new fluorine substituted thiobarbituric acid derivatives (2-4, 7, 8) and their fused/isolated heterobicyclic nitrogen systems (5, 6, 9, 10, 11, 12) have been synthesized from heterocyclization of fluorinated 1,3-diketoamine (1) with CS2 followed by ring closure reactions with primary nitrogen reagents. Structures of the targets have been established from elemental analysis and spectral data. Some synthesized systems have been evaluated as anti-HIV-1 and of cyclin-dependent kinase 2 (CDK2) for cell tumor division.


Introduction
1,3-Diketoamine structural analogues have attracted special interest by virtue of their varied and pharmaceutically useful biological actions as anticonvulsant [1], antianaesthetic [2], anti HIV agents [3].Also, thiobarbituric acids are used to measure the autoxidation of brain homogenates for various animals [4], treate non-alcoholic fatty liver disease [5], and determine formaldehyde and acetaldehyde in the food [6] as well as they form a type of bioactive complex with triphenyltin (IV) [7].The introduction of fluorine atoms to heterocyclic systems often improves their biological properties, due to the stability of C-F formed [8]- [10].Based on these facts, the present work deals with synthetic strategy of some new fluorinated 1,3-diketoamine for building a type of fluorinated thiobarbituric acid derivatives and their fused/isolated heterobicyclic system as anti-HIV agent.

Experimental
Melting points were determined with an electrothermal Bibly Stuart Scientific melting point sample (UK).A Perkins Elmer Model RXI-FT IR system 55529 was used for recording IR spectra of the prepared compounds.A Brucker advance DPX 400 MHz model using TMS as internal standard was used for recording the 1 H and 13 C NMR spectra of the compounds on deuterated CDCl 3 .A GC-MS-GP 1000 Ex model was used for recording the mass spectra of the compounds.Electronic spectra were recorded in ethanol on Shimadzu UV and visible 310 IPC Spectro-photometer.Microanalysis was performed by the microanalytical Center of Cairo University, Egypt.Hexafluorobenzene was used as external standard for 19 F NMR at 8425 MHz (Chemical shift in δ, ppm).
1 H NMR spectrum of 2 exhibited a resonated signals at 2.59 and 3.52 ppm for active CH 2 coupling and enolic protons, with a signals at δ 7.65 -6.98 ppm for aryl protons 13  Fluorinated compound 2 underwent acylation using trifluoroacetic anhydride-trifluoroacetic acid [13] pro-duced5-trifluoroacetyl derivatives 3, while fluorinated aroylation using 4-fluorobenzoyl chloride in warming DMF yielded 5-(4'-fluorobenzoyl) derivative 4 (Scheme 1).Structures of compounds 3 and 4 deduced from spectral measurements.A higher λ max of 4 > 3 > 2 confirm that extension conjugation system was as well as adductive electronic density of COCF 3 and or COC 6 H 4 F. IR spectrum of 4 showed absorption bands at ν 3489 and 3304 attributed to OH, and C=O with 1688, 1660 cm −1 cyclic NCSN in addition at ν 1255 cm −1 for C-F absorption bands.Compounds 3 & 4 showed the lacks of CH 2 . 1 H NMR spectrum of 4 exhibited a resonated signals at 9.71 and 8.71 ppm for OH and aliphatic CH-COCF 3 down field, different aromatic protons at δ 7.75 -7.166 and 7.156 -7.07 ppm. 13C NMR spectrum of 4 recorded signals at δ 172.04, 167.45, 165.17, 164.19 ppm attributed to C=S, and 3C=O carbons.In addition, δ 134.9, 132.2 ppm for C-F and C-N with aromatic carbons at δ 131.8 -122.4 ppm.Morever M/S spectrum of 3 recorded m/z at 372 (M-19) as a base peak, while that of 4 exhibited m/e at 417 (M-44) with a base peak at 379.Fluorinated fused heterobicyclic nitrogen systems 5 and 6 obtained from cyclocondensation of 3 and/or 4 with 4-chlorophenyl hydrazine hydrochloride in refluxing absolute ethanol [14] furnished the fluorinated pyrimido [3,4-d]pyrimidine thiones (5 & 6) respectively (Scheme 1).Structures of compounds 5 and 6 were established from correcteelemental analysis and spectral data.IR Spectrum of 6 recorded an absorption bands at ν 3130, 1710 and 1210 cm −1 for the NH, C=O and C=S functional groups with ν at 1255 for C-F with 1580 cm −1 for C=N functional group. 1 H NMR spectrum of 6 showed a resonated signals at δ 9.55 and 7.8 -7.6 & 7.3 -6.9 reaction through two possible routes (Figure 1).Structure of 8 established mainly from spectral data.UV absorption spectrum showed λ max at 263 nm higher than 2 which is due to new α, β-unsaturated ketonic system formed of 8. 1 H NMR spectrum recorded a resonated signals at δ 9.23 ppm for the arylidene proton.Also, 13 C NMR spectrum of exhibited a different types of carbons at δ 181.10, 165.67, 160.91, 159.76, 159.29, 158.15, and 134.40 -115.17ppm attributed to C=S, C=O, C=N, C-S, C-N and C=C carbons.IR spectrum recorded lacks of OH, NH groups and CH 2 with addition a new group at 1610 cm −1 for CH=C.M/S of 8 showed a molecular ion at m/z 428 (M +2 , 88%), with a base peak at 401.The higher reactivity of compound 8 towards cycloaddition with N-phenyl thiourea to electrophilic carbon at position-3 of thiobarbituric acid followed by addition reaction of H 2 -N-CS group to electrophilic carbon arylidene at posiyion-5 to give 9a.A possible other route is a nucleophilic reaction CS-NH 2 group to a electrophilic carbon at position-3 of thiobarbituric acid followed by addition reaction of HN-Ph group to electrophilic carbons of arylidene at position-5 to give 9a.A possible other route is a nucleophilic reaction CS-NH 2 group to a electrophilic carbon at position-3 of thiobarbituric acid followed by addition reaction of HN-Ph group to electrophilic carbons of arylidene at position-5 to give 9a.The kinetics and mechanism of this reaction were controlled by the highly acidic proton of Ph-NH than H 2 N-CS proton (as thioamide).A higher yield of 9a is due to a high withdrawing of Ph group than amidic CONH 2 .Also, a higher melting point of 9a than 9b is due to a higher stability of 9a than 9b which characterized by a repulsion between phenyl and thiophenegroups.Thus, compounds 9a and 9b are isomeric structure.Structure of compounds 9a & 9b determined from: 1) UV absorption of compounds 9a recorded λ max at 280 nm, and that of 9b at 286 nm; 2) IR absorption spectra of 9a & 9b showed a lacks of OH, NH functional groups and CH 2 ; 3) 1 H NMR spectra of 9a and 9b recorded a resonated signals at δ 7.60 -7.34, 7.29 -7.026 and 7.021 -6.855 ppm for different thiophene and aryl protons; 4) 13   3).Former structure of compound 11 deduced for spectral measurements.A good characterized obtained from that UV spectrum which showed λ max at 256 nm higher than that of 2 (240 nm).IR spectrum recorded a weak band at 3550 cm −1 for OH with 2928, 2858 cm −1 for CH, CH 2 .In addition to 1662 cm −1 attributed to C=O, with ν at 1504, 1438 cm −1 for deformation of CH 2 .Mass spectrum of 11 recorded the molecular ion and a base peak at m/e 720 (M + 25.11), 95 (100).Formation of 12 was deduced from treatment of 2 with piperidine as base to produce the carbanion which attacks the first electrophilic carbon of oxalyl followed by a second attack of other nucleophiliccarbanion of thiobarbituric acid to the second electrophilic carbon of oxalyl chloride (Scheme 4).A possible tautomerism of compound 12 gives us a good indication about the acidic character of two protons within the former structure which facilitated the oxidation  reduction process.
Finally structure of compound 12 established spectral data.UV absorption spectrum showed λ max at 271 nm which is higher than starting material 2. These highly absorption is confirmed that larger extension of heteroconjugation system bearing both bathochromic and hypsochromic moieties.IR spectrum showed a lacks of bands for OH, NH functional groups and CH 2 with the additional bands at 1785, 1680 and 1662 cm −1 for 3C=O groups.A functional groups at ν 1380, 1255 and 1210 cm −1 attributed to NCSN, C=S and C-F functional groups.
1 H NMR spectrum exhibited two resonated signals at δ 9.18 ppm attributed to OH proton from the free solubility of 12 in sodium hydroxide solution. 13

Conclusion
Novel fluorinated thiobarbituric acid derivatives have been synthesized and then evaluated as medicinal agents, Among these compounds 9a > 12 > 2 > 11 exhibited a higher activity as anti HIV agents ,while compounds 12 > 7 > 2 > 3 > 4 have a good activity toward enzymatic inhibition as cyclin-dependent kinase 2 (CDK2) for cell tumer division

Anti-HIV Evaluation
The research for a more effective and less toxic agents has brought into focus potent yet structurally different non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) [19].Abdel-Rahman [20]- [23] synthesized a pool of new fluorinate heterocyclic nitrogen systems as HIV inhibitor agents.Thus, the main aim of the present work tends to search for new fluorinated thiobarbituric acid derivatives and their related fused heterobicyclic nitrogen systems as inhibitors of HIV cases.Some of the synthesized targets have been evaluated as anti-HIV agents.The procedure used in the National Cancer Institute test for agents active against Human Immunodeficiency Virus (HIV) is designed to detect agent acting at any stage of the virus reproductive cycle.The assay basically involves the killing of T 4 lymphocytes by HIV and used the tetrazoliumsalt XTT, as indicator [24].From the results obtained in (Table 1) we showed that, all the fluorinated thiobarbituric acid derivatives exhibited maximum protection <10% for the infected cell and >50% for the uninfected cell.It is interested that, the full fluorinated thiobarbituric acids obtained exhibited maximum protection >20% for the infected cell and >50% for the uninfected cell.Only, the compound 9a recorded a highly percent of protection at lower dose (molar) The indication > specific only partial production of the infected cells at the indicated highest concentration tested.
than other tested compounds.Also, the order reactivities as 9a > 12 > 2 > 11.Based on the resulted data and in compares with results obtained from thiobarbituric acids based HIV-1 integrase inhibitors [3] we can be additional that, the novel fluorinated thiobarbituric acid derivatives in the treatment of HIV infection: 1) nucleoside reverse transcriptase inhibitors; 2) non nucleoside reverse transcriptase inhibitors; 3) protease inhibitors and and fusion inhibitors which led to development of new therapies against the virus especially HIV-1 inhibitors.

Induction of Apoptosis in Human Leukemia Cells
Cancer cells differ from the normal cells in a biochemical processes particularly during the control of cell growth and division.Leukemia is one of the major types of cancer affecting significant segment of the population.An important application of small molecule libraries is the preparation of a directed of focused combinatorial library for assay against specific biological target, fluorine substituted thiobarbituric acids were proven to be biologically very potent and selective.In this study, using the fluorine substituted thiobarbituric acid derivatives as anticancer activity via inhibition of cyclin-dependent kinase 1 (CDK2) especially as inhibitors towards human leukemia cells.These evaluations carried out by applied of standard method [25] in biochemical assay with IC 50 values comparable to olomoucine as control.The result obtained recorded in (Table 2).
From the results obtained in (Table 2) we showed that: detailed anti-cancer (CDK2 for leukemia cells) activity of the fluorinated thiobarbituric acids are very important used as kinase inhibitor activityin the order 12 > 7 > 2 > 3 > 4 > Olomoucine.Preliminary results indicate that many of compounds exhibit an strong effects in compare with the standard used (Olomoucine).The accepting properties of these fluorothiobarbituric acid derivatives are associated with nucleophilicintramolecular substitutions of fluorine atoms, towards the acceptors with cell-cancer.The prominent role of fluorine substituent effects on bioactivity is mainly due to the effect of fluorination of C-H acidity which is predictable and depends on some factors, including the number site of fluorine and the geometry of the conjugate carbanion, which is called bio-conjugation effects.The presented data represent mean values from three independent experiments plus the standard deviation (SD).
C NMR spectrum of 2 showed resonated signals at δ 165.65, 159.63, and 158.02 ppm C=S, 2C=O carbon and δ at 134.52, 134.51 ppm for the C-F carbons, in addition of aromatic carbons.Mass spectrum 2 exhibited the molecular ion with a base peak at m/e 334 and 95.Treatment of ethanolic solution of 2 with FeCl 3 solution gave the deep violet colour which confirms that phenolic formula.Presence of α-active methylene at position-5 of the thiobarbituric acid 2, led to synthesize various fluorinated isolated and/or fused heterobicyclic nitrogen systems.
C NMR spectrum of 12 recorded the resonated signals at δ 181.63, and 168, 166, 162 ppm C=S and 3C=O carbons.Mass spectrum of 12 recorded the m/e at 362 (M +2 ) as a base peak.Finally, synthesized single fluorine which attributed to phenyl ring in all fluorinates systems appeared in the region at δ −125 to −128 ppm.

Table 1 .
The anti-HIV-IC50 values of the fluorinated compounds.