Summary The HIV-1 reverse transcriptase (RT) has two associated activities, DNA polymerase and Ribonuclease H (RNase H), both essential for viral replication and validated drug targets. While all RT inhibitors approved for therapy target the DNA polymerase activity, the search of new RT inhibitors which target the RNase H function and are possibly active on RTs resistant to the known non-nucleoside inhibitors (NNRTI) is a viable approach for anti-HIV drug development. In this report several alizarine derivatives were synthesized and tested on both HIV-1 RT-associated activities. Alizarine analogues K-49 and KNA-53 showed IC50 values for both RT-associated functions around 10 μM. When tested on the K103N RT both derivatives equally inhibited the RT-associated functions, while when tested on the Y181C RT, only KNA-53 inhibited the RNase H function but was inactive on the polymerase function. Mechanism of action studies showed that these derivatives do not intercalate into DNA and do not chelate the divalent cofactor Mg2+. Kinetic studies demonstrated that they are non-competitive inhibitors, they do not bind to the RNase H active site or to the classical NNRTI binding pocket, even though efavirenz binding negatively influenced K-49/KNA-53 binding and vice versa. This behavior suggested that the alizarine derivatives binding site could be close to the NNRTI binding pocket. Docking experiments and molecular dynamic simulation confirmed the experimental data and the ability of these compounds to occupy a binding pocket close to the NNRTI site.

Background: The degradative activity of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), termed ribonuclease H (RNase H), which hydrolyzes the RNA component of the heteroduplex RNA:DNA replication intermediate, is an excellent target for drug discovery. Anthraquinones (AQs) and their derivatives, which are common secondary metabolites occurring in bacteria, fungi, lichens and a large number of families in higher plants, have been reported to have several biological activities including that of inhibiting HIV-1 RT activities in biochemical assays. Methods: We have assayed new AQ derivatives on HIV-1 RNase H activities in biochemical assays. Results: Six series of new AQ derivatives with various substituents at positions 1, 2, 3 and 4 of the AQ ring were tested, and new analogs able to inhibit HIV-1 RT-associated RNase H activity in the low micromolar range were found. Conclusions: Our results demonstrate that AQ derivatives are promising anti-RNase H inhibitors.

The HIV-1 reverse transcriptase (RT) associated ribonuclease H (RNase H) activity hydrolyzes the RNA component of the viral heteroduplex RNA:DNA replication intermediate. Even though this function is essential for viral replication, until now only very few compounds have been reported to inhibit it. Anthraquinones are common secondary metabolites which have diverse biological activities. In particular, some of them have been reported to inhibit the HIV-1 RT polymerase and integrase activities in biochemical assays. Given the structural similarities between integrase and RNase H proteins, we synthesized a series of frangula-emodine derivatives and showed that the introduction of a bromine atom in position 7 of the anthraquinone structure leads to derivatives which are able to inhibit both HIV-1 polymerase and RNase H functions at micromolar concentrations. Mechanism of action studies performed on the 7-brom-6-capital O, Cyrillic-phenacyl-1,8-dihydroxy-3-methyl anthraquinone (K67) showed that this compound is a non-competitive inhibitor of the RNase H function and that it binds to a site which is not overlapping to the non-nucleoside RT inhibitors binding site. This study demonstrates that anthraquinone derivatives may be a scaffold to be further developed to obtain selective HIV-1 RNase H inhibitors and represent a new step toward the identification of new anti-RT agents.

Human immunodeficiency virus 1 (HIV-1) and Hepatitis C virus (HCV) affect 60 and 170 million infected individuals worldwide, respectively, and co-infection by both pathogens is often observed. This represents a serious public health problem that requires the identification of new drugs targeting essential phases of the life cycle of these two viruses. In this report, the synthesis and inhibitory activity of quinizarin derivatives towards both HCV NS5B polymerase and HIV-1 reverse transcriptase associated functions are reported. Our results demonstrate that anthraquinone derivatives are promising anti-polymerase viral inhibitors.

547.673.288Alkylation of 1,2-dihydroxyanthraquinone (alizarin) by α-bromoacetone was studied and its β-acetonyl derivative was chemically modified. The composition and structure of the products were confirmed by elemental analysis; UV, IR, PMR, and 13 C NMR spectroscopy; and mass spectrometry. The synthesized derivatives were tested as inhibitors of HIV-1 RNase H.Key words: alizarin, β-acetonyl derivative of alizarin, HIV-1 RNase H-activity.Anthraquinones are widely distributed in nature [1] and are used in various branches of industry and medicine [2][3][4]. 1,2-Dihydroxyanthraquinone (alizarin, 1) is one of the most common natural hydroxyanthraquinones [5]. It and its methyl, methoxy, and acetoxy derivatives in addition to a glycoside, ruberitrinic acid, have been isolated from various Rubia species and other plants [6,7]. Considering the variety of biological activity of anthraquinone derivatives and the fact that anthraquinone derivatives have been reported as potential HIV-1 inhibitors [8-10], we studied the chemical modification of alizarin and the activity of the resulting derivatives as inhibitors of HIV-1 RNase H.Introduction into 1 of fragments with a carbonyl group enabled a wide range of its polyfunctional derivatives to be prepared and opened pathways for using them in further syntheses. Alkylation of 1 with α-bromoacetone (2), which was prepared by the literature method [11] using brominating agent dioxane dibromide, was used to prepare O-acetonyl derivatives.Reaction of 1 with 2 could form both mono-(3) and di-substituted (4) products depending on the synthesis method because 1 contains two hydroxyls. The reaction of 1 in acetone with added K 2 CO 3 was studied. As shown earlier [12], alkylation of the β-hydroxyl (3) occurred first in all instances with an equimolar ratio of reagents and with an excess of the α-bromoketone. Formation of product 4 was not observed with an equimolar ratio of reagents. Performing the reaction with a two-fold excess of the bromide and increasing the time (to 78 h) formed a mixture consisting of the mono-and di-substituted derivatives (3 and 4), which were separated by column chromatography over silica gel. Formation of the mono-and disubstituted products is easily followed by TLC using the color of the compounds (3 is yellow whereas 4 is light yellow), by chromatographic mobility, and by the presence or absence of the characteristic color from the Borntreger reaction (ammonia vapor). The maximum yield of 4 was 54% for the reaction with a three-fold excess of the bromide for 60 h.The purity of the products was confirmed by TLC. They were identified using elemental analysis and UV, IR, PMR, 13 C NMR, and mass spectra. They were crystalline compounds that were very soluble in polar organic solvents and insoluble in water.