Synthesis and in vitro Anti-HIV Screening of Certain 2-(Benzoxazol-2-ylamino)- 3H-4-oxopyrimidines

Sherif A. F. ~ostom'*, Hesharn T. Y. F*, Manal N. S. saudi3 1 Division of Medicinal Chemistry, Faculty of Medicine and Allied Sciences, King Abdul-Aziz University, P.O. Box 80205, Jeddah 2 1589, Kingdom Saudi Arabia. 2 School of Pharmacy, University of Mississippi, Mississippi 38677, USA 3 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University, 2 12 15 Alexandria, A. R. Egypt.


Introduction
The discovery of human irnmunodefficiency virus 1 (HIV-1) as the causative agent of the acquired immunodefficiency syndrome (AIDS) in 1983 ' stimulated an unprecedented level of research activity directed towards both the prevention and treatment of this debilitating lethal disease.Despite the international efforts to control HIV 1 AIDS pandemic through behavioral modifications and other interventions, more than 15,000 people become infected every day, 95 % of whom live in developing countries 233.Information about the genornic structure and replication cycle of retroviruses has facilitated the identification of the biochemical targets for attack by potential therapeutic agents for the treatment of HIV infection.Many of these targets are key proteins involved in the HIV replication cycle.These include formation of proviral DNA by the reverse transcriptase enzyme (RT), integration of proviral DNA into the host DNA by the integrase enzyme, and cleavage of the precursor viral proteins by the protease enzyme ' .In recent years, particular attention has been focussed on the biological significance of the virally encoded RT enzyme, which mediates the conversion of viral RNA genome to proviral DNA I.Therefore, clinically relevant agents which have been successfblly developed are the RT inhibitors, which in their turn are classified into two main categories: nucleoside RT inhibitors (NRTIs) and nonnucleoside RT inhibitors (NNRTIs).NRTIs such as 3'-azidothymidine (AZT) and dideoxyinosine (dd1) act by competitive inhibition of HIV RT through incorporation into the growing viral DNA chain and cause chain termination.They require intracytoplasmic activation by cellular enzyme to the triphosphate form 6 .They are now used clinically for the treatment of HIV infections, however, their use is limited by their significant toxicities 7. On the other hand, NNRTIs are a diverse group of compounds that share a number of common biochemical and pharmacological properties.
Recently, De Clercq * has reported that at least thirty different classes of NNRTIs are available.
Unlike the nucleoside antimetabolites, the NNRTIs do not require bioactivation.They block the HIV RT reaction through interaction with an allosterically located non-substrate binding site by a non competitive mechanism.When bound into their pocket at the HIV RT, the NNRTIs maintain a very similar conformational shape.They roughly overlay each other in the binding pocket and appear to knction as n-electron donors to aromatic side chain residues surrounding the pocket 9.
NNRTIs are extremely potent and selective as they do not inhibit RT of other retroviruses including HIV-2.They have high therapeutic indices (in contrast to nucleosides) and do not inhibit mamalian DNA polymerases lo.As NNRTIs interact with a specific binding site on the enzyme, any slight variation brought about by a single point mutation can have a significant impact on the sensitivity of the virus towards members of this group, and high-level resistance can develop quickly ' I .Among the already reported distinctive NNRTIs, 3-[(4,7-dichlorobenzoxazol-2-yl)-methyl~no]-5-ethyl-6methylpyridin-2(1H)-one; 0, 697, 661) was reported to inhibit the spread of the HZV infection by 95 % in MT4 cell culture.On basis of potency, selectivity, oral bioavailability and appropriate safety and tolerability studies, this lead compound was selected for phase 1 clinical trials to determine these parameters in man 12.
The pre-mentioned considerations highlight the importance and the urgent need to continue searching for new more potent, less toxic and more selective anti-HIV agent.In this view, and as a result of the recent growing interest of benzoxazoles as chemotherapeutic agents ')-I8, it was designed to synthesize and investigate the anti-HIV activity of some new 2-(benzoxazol-2-y1amino)-3H-4-oxopyrimidines.The newly synthesized compounds are structurally related to the lead compound (L 697, 661) which constitutes the benzoxazole ring linked at Cz to a pyridone moiety through a two atom spacer.The target compounds were patterned so as to comprise the benzoxazole and the bioisosteric pyrimidinone counterparts separated by NH linker.In addition, the pyrimidinone moiety is considered as an essential component in many nucleoside and nonnucleoside antiviral agents 19"'.
The substitution pattern of the pyrimidinone ring was carefblly selected so as to confer different electronic environment to the molecule and believed to be responsible for such biological activity in structurally relevant compounds such as NSC 666268 22.
It was also attempted to investigate the previously reported, structurally related compound 2-

Chemistry
The synthetic strategies adopted to obtain the target compounds are depicted in schemes 1 and 2.
On the other hand, the remaining compounds failed to counteract the cytopathic effect of HIV, since the cell growth of HIV-infected cells lied between 5.14-44.06% (Table 1).Values of cell growth of HIV-infected cells between 0-50% indicated lack of any substantial anti-HIV activity.
However, all compounds failed to suppress cell proliferation of uninfected CEM cells at micromolar Owing to the weak anti-HIV activity displayed by the newly synthesized compounds, it was difficult to establish a relationship between the substitution pattern of the pyrimidine ring at Cd, C5, Cg and the biological activity.However, the anti-HIV profile of compound 8 would encourage fbrther structural modifications.A solution of 2-guanidinobenzoxazole 1 (0.5 g, 3 mmol) 24 and 2-actylbutyro-lactone (0.8 g, 6 rnrnol) in bromobenzene (10 rnl) was refluxed for 5 h.After cooling to room temperature, the separated solid product was filtered, washed with cold ethanol, dried and recrystallized.
After being cooled to room temperature, the yellowish solid product was separated, suspended in water and acidified with dilute hydrochloric acid till pH 3-4.

B. In vitro anti-HIV screening
The in vitro drug testing system was performed in the National Cancer Institute's Developmental Therapeutics Program (DTP), AIDS antiviral screening program, according to a reported The assay involved killing of T4 lymphocytes by HIV.T4 lymphocytes (CEM cell line) were exposed to HIV at a virus-to-cell ratio approximately 0.05 and treated with the test compounds, dissolved in dimethyl sulfoxide, at doses ranging from lo4 to lo4 M. A complete cycle of virus replication is necessary to obtain the required cell killing (incubation at 3 7 ' ~ in a 5% Synthesis and in vitroAnti-HIV Screening of Certain 2-(Benzoxazol-2-ylamino)-3H-4-... 59 NSC 666268

Table 1 .
Reduction of in vitro HIV-induced cytopathic effect (% protection) and cytotoxic dose (IC50, pM) of compounds 2which rnaxjmum reduction of viral cytopathic effect occurred b SE denotes the standard error (n = 4) c All the data are significantly different &om the control (Student t-test, p > 0.001) d ECso = 12.1 pM; TI = 5.62 e ECso = 6.04 pM; TI = 6.14 f Not mentioned g EC50=>0.00257pM;TI>3.89 x concentrations although remarkable inhibition of cell growth was observed at much higher concentrations.Synthesis and in vitro Anti-HIV Screening of Certain 2-(Benzoxazol-2-ylamino)-3H-4-.. .65 Referring to the results recorded in table 1, one can notice that, substitution of Cg of 2-(benzoxazol-2-vlamino)-6-hydroxy-3H-4-oxopdine 8 with alkyl (aryl) or arylazo groups (as in 10a-c and l l b ) resulted in sigruficant reduction in the anti-HIV activity (percent of protection 12.31-40.87%).Conversion of the same derivative to the dichloro analog 9 led to almost abolishment of the biological activity (percent of protection 5.14 %).

a
Melting points were determined in open-glass capillaries on a Stuart melting point apparatus and were uncorrected.The idkared (IR) spectra were recorded on 470-Shimadm infrared spectrophotometer using the KBr disc technique.The 'H-NMR-(6-ppm) spectra were recorded on a Bruker (400 MHz) spectrometer using tetrarnethylsilane as the internal standard and DMSO-d6 as the solvent.Splitting patterns were designated as follows: s; singlet; d: doublet; m: multiplet.Mass spectra were recorded on a Finnigan SSQ 7000 GC-MS, ionization energy 70 eV.Elemental analyses were performed at the Microanalytical Unit, Faculty of Science, Cairo University, Cairo, Egypt, 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 at h Table 2. Physicochernical and analytical data of compounds 2Analyzed for C,H,N,S; results are within -;t 0.4 % of the theoretical values for the formulae given 2-(Benzoxazol-2-ylamino)-5-(2-hydroxyethyl)-6-methyl-3H-4-oxopyrimidine (2)