Synthesis and Biological Evaluation of 2-Thioxopyrimidin-4(1H)-one Derivatives as Potential Non-Nucleoside HIV-1 Reverse Transcriptase Inhibitors

A series of new 5-allyl-6-benzylpyrimidin-4(3H)-ones bearing different substituents at the C-2 position of the pyrimidine core have been synthesized and evaluated for their in vitro activities against human immunodeficiency virus type 1 (HIV-1) in the human T-lymphotropic type (MT-4 cell cultures). The majority of the title compounds showed moderate to good activities against HIV-1. Amongst them, 5-allyl-6-benzyl-2-(3-hydroxypropylthio)pyrimidin-4(3H)-one analogue 11c exhibited the most potent anti-HIV-1 activity (IC50 0.32 µM). The biological testing results clearly indicated that the substitution at C-2 position of the pyrimidine ring could increase the anti-HIV-1 reverse transcriptase (RT) activity.


Introduction
The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1 RT) is one of the main targets of drugs used in the treatment of AIDS [1,2]. Several RT inhibitors have been developed and approved by the FDA and are currently in clinical use. Among several kinds of anti-HIV agents, non-nucleoside reverse transcriptase inhibitors (NNRTIs), specifically targeting to reverse transcriptase, have attracted wide attention due to their high specificity, excellent potency and low cytotoxicity [3,4]. NNRTIs are a class of antiretroviral drugs which deactivate HIV-1 reverse transcriptase by inducing conformational change of the enzyme via binding to a hydrophobic site close (approximately 10 Å) to its catalytic site [5]. Since these compounds do not need intracellular metabolic activation, have relatively low cytotoxicity, act on a nanomolar scale and are not active against other retroviruses, they are highly specific HIV-1 inhibitors. Among NNRTIs, dihydro alkoxy benzyl oxopyrimdines (DABOs) are an interesting class of compounds active at nanomolar concentration. This class was first reported in 1992 and further developed during the following years into S-DABOs, N-DABOs and related analogues . Over the past decade, 2-alkylthio-6-benzylpyrimidin-4(3H)-ones (S-DABOs) have been the subject of great interest and have led to the identification of several new structures [27][28][29] displaying excellent activities as non-nucleoside inhibitors of HIV-1 reverse transcriptase (RT). In our continuing efforts to find novel effective and selective anti-HIV-1 agents in 2-alkylsulfanyl-6-benzyl-3,4-dihydropyrimidin-4(3H)-ones (S-DABOs) type NNRTIs [30][31][32][33], herein we present the synthesis and anti-HIV-1 activities of a series of S-DABO analogs.

Pharmacological Screening
The anti-HIV activities and cytotoxicity's of the synthesized S-DABOs analogues are summarized in Table 1. The expression of HIV-1 was quantified by two different methods, either the HIV-1 antigen detection assay enzyme-linked immunosorbent assay (ELISA) [34] or indirectly by the 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay [35]. In general, the activity of the tested compounds including S-DABOs derivatives in the side chains at C-2 is to be expected when compared to those previously reported for S-DABOs. As illustrated in Table 1, we could see that most of those compounds showed moderate to potent inhibition of RT activity, suggesting that HIV-1 RT was the target of this series of S-DABO derivatives. The activity and cytotoxicity of these newly synthesized S-DABO analogues were evaluated in MT-4 cells for their ability to inhibit HIV-1 in MT-4 cells,in comparison with (6-benzyl-1-(ethoxymethyl)-5-isopropylpyrimidine-2,4-dione (MKC-442) and azidothymidine (AZT) used as reference drugs. Most of the tested compounds exhibited moderate to good activities against wild-type HIV-1 with EC50 values ranged from 35.00 to 0.32 µM. The most active compound 11c showed the highest activity against wild-type HIV-1 with an EC50 value of 0.32 µM. The other compounds displayed moderate activity 2e, 2j, EC50 2.5 and 2.60 µM, respectively or were inactive 2c, 2k, 3b, 4 and 9, against HIV-1 replication.

Chemistry
All melting points were uncorrected and determined on a Büchi melting point apparatus (Hamburg, Germany). NMR spectra were recorded on a Varian Gemini 2000 NMR spectrometer (Varian Inc., Palo Alto, CA, USA) at 300 MHz for 1 H NMR and 75 MHz for 13 C NMR using tetramethylsilane (TMS) as internal standard. All chemical shifts are quoted in δ values using parts per million scale (ppm) downfield from TMS at the University of Southern Denmark, Denmark. EI mass spectra were recorded on a Finnigan MAT SSQ 710 spectrometer (Finnigan MAT, Madison, WI, USA) at 70 eV at the University of Southern Denmark, Denmark. Elemental analyses were performed at Atlantic Micro lab, Inc., Atlanta, GA, USA, and the found values agreed favourably with the calculated ones. The progress of reactions was monitored by TLC (analytical silica gel plates 60 F254). Merck silica gel (0.040-0.063 mm) was used for column chromatography. The analytical spectral analyses were supported by Erik B. Pedersen, University of Southern Denmark, Institute of Chemistry, and antiviral screening in vitro through the Retrovirus Laboratory, Department of Virology, State Serum Institute, Copenhagen, Denmark.

5-Allyl 6-(benzyl)-2-(methylthio)pyrimidin-4(3H)-one (2a).
A mixture of compound 1 (1.29 mmol), 10 mL hexamethyldisilazane (HMDS) and 10 mg (NH4)2SO4 were refluxed for 2 h. The remaining HMDS was removed under reduced pressure. 1 mL of anhydrous DMF and (2.24 mmol) of methyl iodide were added and the solution was stirred at room temperature for 5 h. 100 mL of dichloromethane was added and the solution was washed with 20 mL anhydrous NaHCO3. The organic layer was dried over Na2SO4 and evaporated till dryness under reduced pressure. The solid was washed with benzene to remove iodide and the solid was dissolved in CH2Cl2, dried and purified by column chromatography (

Virus and cells
The HIV-1 strains HTLV-IIIB and NNRTI resistant strain N 119 were propagated in H9 cells at 37 °C, 5% CO2 using RPMI 1640 with 10% heat-inactivated fetal calf serum (FCS) and antibiotics (growth medium). The culture supernatant was filtered (0.45 nm), liquated and stored at −80 °C until use. Both HIV-1 strains were obtained from the NIH AIDS Research and Reference program.

Inhibition of Human Immunodeficiency Virus Type 1 (HIV-1) Replication
Compounds were examined for possible antiviral activity against both strains of HIV-1 using MT-4 cells as target cells. MT4 cells were incubated with virus (0.005 MOI) and growth medium containing the test dilutions of compounds for six days in parallel with virus-infected and uninfected control cultures without compound added. Expression of HIV in the cultures was quantitated by the HIV-1 antigen detection assay ELISA [27] or indirectly quantified using the MTT assay [28]. Compounds mediating less than 30% reduction of HIV expression were considered without biological activity. Compounds were tested in parallel for cytotoxic effect in uninfected MT-4 cultures containing the test dilutions of compound as described above. A 30% inhibition of cell growth relative to control cultures was considered significant. The 50% inhibitory concentrations (IC50) and the 50% cytotoxic concentrations (CC50) were determined by interpolation from the plots of percent inhibition vs. concentration of compound.

Conclusions
Non-nucleoside reverse transcriptase inhibitors (NNRTI) are key components in highly active antiretroviral therapy for treating HIV-1. Herein we present the synthesis for a series of S-DABOs analogues and evaluated as non-nucleoside HIV-1 reverse transcriptase inhibitors for inhibition of HIV-1 replication. Various chemical and spectral data supported the structures of the newly synthesized compounds. The majority of the tested compounds showed moderate to good activities against HIV-1 with an IC50 range from 35.60 to 0.32 µM. These results provided useful indicators for guiding the further rational design of new S-DABO analogues as more active and selective HIV-1 inhibitors.