Synthesis and anti-HIV activity evaluation of new phenyl ethyl thiourea (PET) derivatives

This manuscript describes the synthesis of a new series of phenyl ethyl thiourea (PET) derivatives, with the aim to extend the SAR studies of the well known PET molecules endowed with anti-HIV activity. Preliminary results indicated that the synthesized compounds possess low anti-HIV activity


Introduction
Human immunodeficiency virus type-1 (HIV-1) is the causative agent for the transmission and development of the acquired immunodeficiency syndrome (AIDS).AIDS remains one of the most urgent world health problems, being the leading cause of death in Africa and the fourth worldwide. 1ven if there is no definitive cure for HIV infection, a number of drugs slow or halt disease progression.However, HIV can rapidly become resistant to any single antiretroviral drug, therefore a combination of three or more drugs are usually required to effectively suppress the virus.The highly active antiretroviral therapy (HAART) 2 consists of the combination of nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs) with non-nucleoside reverse transcriptase inhibitors (NNRTIs) or protease inhibitors (PIs). 2 In this context, while NRTIs (e.g.zidovudine, lamivudine, abacavir, tenofovir) act competitively at the catalytic site of the RT as DNA chain-terminating analogues of the natural deoxynucleoside triphosphates, NNRTIs (e.g.nevirapine, efavirenz) bind to an allosteric site located about 10 Å from the catalytic site thus leading to a noncompetitive inhibition of the enzyme. 3The latter ones, despite their lower toxicity with respect to NRTIs, are particularly vulnerable to the development of viral resistance caused by mutations in RT that can retain viable enzymatic function.Therefore significant efforts in this field have focused on developing new NNRTIs with a favourable profile of resilience to many drug resistant mutations. 46] This compound showed a significant inhibition value against the RT.
Basic SAR studies were performed by notionally dividing LY73497 into four portions and independently varying each one of them. 5The results of these SAR studies provided evidence of important features for the interaction with RT: i) small electron withdrawing substituents (F, Cl) can be favorably introduced on the phenyl ring especially in ortho position; ii) the ethyl linker is optimal for the activity; iii) the urea derivatives are less active than the corresponding thiourea derivatives; iv) the thiazolyl nucleus can be replaced successfully with other heterocyclic rings such as pyridine, pyrazine, benzothiazole, imidazole, triazole.
The aim of this work was to synthesize new PET derivatives 1 (Figure 2), in which important requirements for the anti-HIV activity such as two aromatic systems, benzene and pyridine, and the thiourea moiety were maintained, while the ethylene bridge was modified by introducing a carbonyl group and a hydroxyl function.This modification took into account the work of Högberg et al. 7 in which the introduction of a carbonyl group in proximity of the phenyl ring resulted in an enhanced the anti-HIV activity both in enzymatic assays and in cell cultures.The importance of this substitution on the ethyl linker was recently confirmed by QSAR studies. 8n compounds of series 1 the introduction of a hydroxyl group on the ethyl linker should reduce the conformational freedom by forming an intramolecular hydrogen bond with the sulfur atom.Thus, a series of ethers (series 2, Figure 2) were also synthesized to compare them with derivatives of series 1 and confirm our hypothesis.On the aromatic ring various halogens atoms were introduced, such as chlorine and fluorine, in agreement with the SAR studies on PET derivatives.

Results and Discussion
The synthesis of N 1 -(1-hydroxy-2-oxo-2-phenylethyl)-N 3 -pyridin-2-yl-thioureas 1a-f was realized by nucleophilic addition of 2-pyridylthiourea 5 to the variously substituted 2-oxophenylacetaldehydes 4a-f (Scheme 1), prepared by easy oxidation of the corresponding acetophenones 3a-f with selenium dioxide according to a previously reported procedure. 9The reaction between 4a-f and 5 has been promoted by catalytic amounts of the mixture acetic acid/hydrochloric acid in dioxane.By maintaining unchanged the reaction conditions and switching the solvent from dioxane to ethanol, it was possible to obtain N 1 -(1-ethoxy-2-oxo-2-phenylethyl)-N 3 -pyridin-2-ylthioureas 2a-f, realistically due to the addition of ethanol to the N-aroylmethylene thiourea intermediate.Both series 1a-f and 2a-f have been tested to evaluate their anti-HIV activity at National Cancer Institute (NCI) of Bethesda, USA.The assay was carried on MT-4 cell lines exposed to the strain HIV-1 III B in the presence of compounds 1a-f and 2a-f to determine the concentration that reduces to the 50% (EC 50 ) the cytopathic effect induced by HIV-1 in MT-4 cells, the cytotoxicity (CC 50 ) and the selectivity index that is the ratio CC 50 /EC 50 .
Only the derivatives 1a, 1c, 2a and 2c showed a moderate anti-HIV activity (Table 1), with an EC 50 value of 118.08 µM and 166.25 µM for the hydroxyl derivatives 1a and 1c, and of 67.05 µM and 69.48 µM for the O-ethyl derivatives 2a and 2c respectively.All the other compounds protected the MT-4 cells from the cytopathic effect in a low percentage (8-22%).Generally, all compounds showed a very low cytotoxicity profile >400 µM for the derivatives 1a-f and >200 µM for O-ethyl analogues 2a-f.The obtained results suggested that the presence of a chlorine atom at position 2 or 4 of the phenyl ring positively affected the anti-HIV activity of this new series of PET derivatives.The slightly higher activity of O-ethyl derivatives 2a and 2c with respect to the corresponding hydroxyl derivatives 1a and 1c indicated that a hydrophobic character of the linker is favourable for antiviral activity.This is confirmed when we compare our results either with the LY73497 analogues or the series of compounds with the carbonyl function next to phenyl ring. 5,7n conclusion, this work may offer an additional contribution to extend the SAR studies of the well known PET derivatives endowed with anti-HIV activity, useful for further developments of this series.

Experimental Section
General.Melting points were determined on a Kofler hot-stage apparatus and are uncorrected.Elemental analyses (C, H, N) were carried out on a Carlo Erba Model 1106 elemental analyzer; the results were correct within ±0.4% of the calc.values.Merck silica-gel 60 F 254 plates were used for anal.TLC, and column chromatography (CC) was performed on Merck silica gel 60 (70-230 mesh). 1 H-NMR Spectra were recorded in CDCl 3 on a Varian Gemini-300 spectrometer; chemical shifts δ in ppm rel. to Me 4 Si, coupling constants J in Hz.

General procedure for the synthesis of substituted 2-oxophenyl-acetaldehydes 4
Selenium dioxide (11 mmol) was solubilized in a mixture water/dioxane (26 mL, 4/96) at 60°C, when the solution was homogenous, it was cooled and the required acetophenone 3 (11 mmol) was added.The reaction mixture was heated at 100°C for 12 h filtered on celite and then evaporated to dryness.The residue was diluted with ethyl acetate, washed with water, then saturated NaHCO 3 and dried (Na 2 SO 4 ).The solvent was evaporated and the residue containing the title compound in quantitative yields (as observed by TLC) was used for the next step without any further purification.

General procedure for the synthesis of N 1 -[2-aryl-1-hydroxy-2-oxoethyl]-N 3 -pyridin-2-ylthioureas 1a-f
2-Pyridylthiourea 5 (200 mg, 1.3 mmol), acetic acid (0.6 mL) and hydrochloric acid (0.6 mL) were added to a solution of variously substituted 2-oxophenyl-acetaldehydes 4a-f 9 (1.5 mmol) in 15 mL of dioxane.The reaction mixture was stirred at 100 °C for 8 h, then cooled and evaporated to dryness.The residue was purified by column chromatography using silica gel as absorbent with 9:1 CHCl 3 /MeOH as eluent to give the title compounds.Pharmacology.The in vitro anti-HIV drug testing system was performed in the National Cancer Institute's Developmental Therapeutics Program, AIDS antiviral screening program, according to reported procedures. 10The assay involves the killing of T 4 lymphocytes by HIV-1.T 4 lymphocytes (CEM-SS cell line) are exposed to HIV-1 (RF strain) at a multiplicity of infection (MOI) of approximately 0.05.Each agent, dissolved in dimethyl sulfoxide, was added at varying concentrations ranging from 10 -9 to 10 -5 M. Uninfected cells with the test compound serve as toxicity control, and infected and uninfected cells without the compound serve as basic controls.Activity is expressed as the effective concentration 50% (EC 50 ) which represents the concentration of each compound resulting in a 50% reduction of the viral cytopathic effect.The 50% inhibitory concentration (CC 50 ) represents the toxic concentration of drug resulting in 50% growth inhibition of normal, uninfected cells.The selectivity index (SI) is determined by dividing CC 50 by EC 50 .
bConcentration that reduces to the 50% the growth of the MT-4 cells.c Selectivity index: ratio CC 50 /EC 50 .