Vol. 54 No. 4/2007, 863–868 Communication on-line at: www.actabp.pl Synthesis and anti-HIV properties of novel

Novel 6-phenylselenenyl-5-propyluracils were synthesized from 5-propyluracil with the use of regioselective synthesis to give 1-[(2-hydroxyethoxy)-methyl]-6-phenylselenenyl-5-propyluracil (6), 1-ethoxymethyl-6-phenylselenenyl-5-propyluracil (9) and 1-benzyloxymethyl-6-phenylselenenyl-5-propyluracil (10). Interaction of these compounds with recombinant HIV-1 reverse transcriptase (RT) was evaluated using a non-isotopic colorimetric method. Compounds 9 and 10 exerted potent HIV RT inhibition (IC(50) 0.06 and 0.05 microM respectively) while compound 6 showed moderate inhibition (IC(50) = 3.5 microM). Potent anti-HIV-1 activity in MT-2 cells inoculated by a syncythia-inducing HIV-1 (cat #3 strain) laboratory isolate was exerted by compounds 9 and 10 (EC(50) 0.62 microM and 0.025 microM, respectively), while compound 6 showed only moderate activity (IC(50) = 4.1 microM). In addition, compound 10 showed very good in vitro therapeutic index (TI > 2046), indicating that it is a potential anti-HIV/AIDS drug.


INTRoDuCTIoN
Human immunodeficiency virus (HIV), the etiological agent of AIDS, encodes RNA-dependent DNA polymerase (reverse transcriptase, RT) but not the specific enzymes required for the phosphorylation of 2',3'-dideoxypyrimidine and purine nucleosides or their phosphonates (De Clercq, 1995).To exert antiviral activity these analogues must undergo three-or two-step phosphorylation by host cell kinases and/or be metabolized by other metabolizing enzymes.The phosphorylated derivatives act as RT competitive inhibitors and/or DNA chain terminators (Vandamme et al., 1998).The previously discovered 6-arylthio-and 6-arylselenenyl acyclonucleosides (Tanaka et al., 1991;Goudgaon & Schinazi, 1991) present a new class of potent and highly selective anti-HIV-1 agents which act as non competitive inhibitors of HIV RT and bind allosterically to its hydrophobic pocket close to the catalytic site, distorting the conformation of RT to the point where it cannot function enzymatically (Pan et al., 1994;Vandamme et al., 1998).While 6-arylthio acyclonucleosides inhibit only HIV-1, some 6-phenylselenenyl analogues are active also against HIV-2.5-Alkyl-6-selenenyl acyclonucleosides have been little studied, however, 1-ethoxymethyl-6-phenylselenenyl-5-ethyluracil is a promising candidate for AIDS chemotherapy (Ni et al., 1995).To enhance the hydrophobic properties of the above-mentioned compounds, and therefore the penetration to the central nervous system, we decided to synthesize and investigate their novel 5-propyl analogues.

Biology
Determination of HIV-1 reverse transcriptase activity.The activity of HIV-1 reverse transcriptase (RT) was determined using a non-isotopic colorimetric method (Eberle & Knopf, 1996; Roche Biomolecu-lars) involving incorporation of digoxigenin and biotin-labelled dUTP into DNA (Roche Biomoleculars procedure and KIT No. 1468120).
Cell toxicity.The cytotoxicity of investigated compounds was determined in an established MT-2 cell line.The cells were grown in medium enriched with known different concentrations of the tested compounds.After 3, 7, 10, 14 days the viability of cells in relation to control culture was estimated by a commonly used colorimetric 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrozolium bromide (MTT) method (Mossman, 1983), and CC 50 was calculated.
Anti-HIV activity in MT-2 cell cultures.Identical numbers of MT-2 cells (10 6 /mL) were incubated for 24 h in RPMI medium supplemented with 10% foetal calf serum (FCS), enriched with known amounts of the tested substances in standard conditions (37 o C, 5% CO 2 in a humid environment).The cell cultures were inoculated with a syncytia-inducing HIV-1 (cat #3 strain) laboratory isolate.After 4 h of inoculation the cells were washed 5 times with RPMI and unattached virions were removed.The cells were resuspended in medium enriched with the tested substances and 200 µL of cell suspension was placed into a 96 cell culture plate (2 × 10 5 cells/well).For each concentration of the tested substances cell cultures were done in triplicate.A positive control contained an identical concentration of HIV-1 inoculated cells maintained in RPMI and 10% FCS only.Every three days half of the culture medium (100 µL) was collected and replaced with an identical amount of fresh one.The collected media were frozen.The cells were cultured for up to 14 days.HIV replication and the inhibitory effect of the substances under investigation was estimated by measuring the amount of p24 protein in collected media.The efficacy of HIV-1 replication inhibition exerted by the tested compounds was related to controls cultured in medium without the tested compounds as well as with cells cultured in medium enriched with known amounts of AZT.The IC 50 and IC 90 values were calculated.

Syntheses
Several approaches lead to the synthesis of 1-[(2-hydroxyethoxy)-methyl]-5-alkyluracils.Three most convenient methods were evaluated.Two of them are based on the condensation of corresponding bis(tri-methylsilyl)uracils with acetoxymethyl ether or chloromethyl ether catalysed by SnCl 4 and the yields obtained are moderate to good (38-72%).However, the "one-pot" modification reported by Ubasawa et al. (1995) afforded acyclopyrimidine nucleosides in better yields (63 to 88%).This method seemed to be the most convenient and after certain modifications was applied to the synthesis of the 5propyluracil derivatives.
5-Propylacyclouridine derivatives 7 and 8 were prepared by Bu 4 NI-promoted alkylation of the di-O-TMS derivative of 5-propyluracil with the corresponding ethyl and benzyl chloromethyl ether.

Structure-activity relationship
Previously it was reported that 1-benzyloxymethyl-6-phenylselenenyl-5-ethyluracil showed considerable in vitro anti-HIV-1 activity with EC 50 = 0.017 µM in MT-2 cells.This analogue was also a potent inhibitor of recombinant HIV-1 reverse transcriptase (IC 50 = 8 nM) (Pan et al., 1994).However, its in vitro therapeutic index (TI) was as low as 250, disqualifying this compound as a potential anti-HIV/AIDS agent.Therefore it was of interest to check whether enhancing of the hydrophobic properties of this compound by replacing 5-ethyl in the pyrimidine ring with the more hydrophobic 5-propyl substituent would decrease the cytotoxicity and increase the therapeutic index.
It can be seen that a high antiviral activity was exerted only by those 5-propyl-6-phenylselenenyl acyclonucleosides which at the end of the acyclic moiety contained additional hydrophobic groups, such as ethoxymethyl (compound 9) or benzyloxymethyl (compound 10), while compound 6 containing at this place the hydrophilic hydroxyl group exerted only a moderate activity.Of special interest is the significantly higher in vitro therapeutic index (TI > 2046) of compound 10 as compared to the above-mentioned 1-benzyloxymethyl-6-phenylselenenyl-5-ethyluracil (TI = 250).
To explain the mode of action of these compounds, their interaction with recombinant HIV-1 reverse transcriptase was investigated (Table 2).Compounds 9 and 10 were potent allosteric inhibitors of this enzyme and the data obtained point to a high correlation of HIV RT inhibition with antiviral activity.

Table 1 . Anti-HIV activity and cytotoxicity of 6-phenylselenenyl-5-propyluracils in MT-2 cells
Pan et al. (1994)ration of the compound required to reduce viral cytopathogenic effect by 50%; b Effective concentration of the compound required to reduce viral cytopathogenic effect by 90%; c Cytotoxic concentration of compound required to reduce viability of MT-2 cells by 50%; d Therapeutic index, TI = CC 50 /EC 50 ; ePan et al. (1994) a