Design and Synthesis of WM 5 Analogues as HIV-1 TAR RNA Binders

Department of Pharmaceutical Sciences, University of Perugia, 06123 Perugia, Italy Department of Chemistry, Biology and Biotechnology, University of Perugia, 06123 Perugia, Italy Department of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131, Padova, Italy Rega Institute for Medical Research, Laboratory of Virology and Chemotherapy, K.U. Leuven, K.U. Leuven, B-3000 Leuven, Belgium


INTRODUCTION
In the past decades, considerable work has been done in exploiting small non-coding hairpin RNA fragments as drug targets.However, compared with the compounds targeting proteins, the identification of molecules that selectively bind to structured RNA motifs remains an important challenge [1 -3].One of the most studied RNA structures is the Transactivation response element (TAR) of HIV-1 genome [4 -9], a short stem-bulge-loop structure located in the long terminal repeat Numerous derivatives have been designed and synthesized starting from WM5 with the aim to gain Structure-Activity Relationship (SAR) insights, reduce the cytotoxicity and further investigate the molecular mechanism of action [21].As a consequence, many compounds endowed with good HIV-1 and HIV-2 inhibitory activity in both chronically infected and acutely infected cells have already been identified.However, a direct correlation between the anti-HIV activity and their ability to interfere with the Tat-TAR RNA complex was not always observed [22].On the other hand, when the quinolone nucleus lacking both C-7 arylpiperazine and C-3 carboxylic functions was properly functionalized with protonable groups at the C-2 position, a series of 2-phenylquinolones was obtained showing an improved ability to displace Tat-TAR complex but at the expense of antiviral activity [23,24].
Here, we came back to WM5 preparing a series of analogues designed to enhance its ability to recognize the viral nucleic acid TAR RNA.Considering that TAR plays its essential role in the Tat-mediated transcription by specifically binding Tat and that in the Tat-TAR complex the arginine rich basic portion of Tat is responsible for the TAR binding, we decided to modify WM5 by inserting guanidine or amidine groups as well as other protonable moieties intended to bind electrostatically the phosphate backbone of TAR.In the present paper, the synthesis of a series of WM5 analogues modified at N-1, C-6 or C-7 position Fig. ( 1), and their anti-HIV-1 activity along with their cytotoxicity in MT-4 cells, are reported.The active compounds were also evaluated in a Fluorescence Quenching Assay (FQA) to determine their ability to interfere with the formation of the complex between TAR RNA and a truncated Tat peptide.

Design of WM5 Analogues
Starting from WM5, the C-6 amino group was replaced by a more basic guanidine in compound 1, while at C-7 position an amidine moiety replaced the 2-pyridine ring in compound 2, and an imidazoline ring was present at the C-4 position of the pyridine in compound 3 Fig.(1).Pyridine and (pyridinyl)piperazine moieties were inserted at the N-1 position through methyl, ethyl, or propyl chains, as in compounds 4, 5 [25] and 6, respectively Fig. (1).Moreover, by maintaining the propyl chain, the piperazine and the pyridine rings were alternatively deleted as in compounds 7 and 8, respectively Fig.

Chemistry
Starting materials, reagents, and solvents that were commercially available were used as supplied.The reactions were monitored by TLC on silica gel 60F254 (Merck) and the compounds were visualized by UV and/or iodine.Flash chromatography columns were performed on Merck silica gel 60 (mesh 230-400).After extraction, organic solutions were dried using anhydrous Na 2 SO 4 , filtered, and evaporated to dryness at reduced pressure using a Büchi rotary evaporator.Yields are of pure products and were not optimized.Melting points were determined in capillary tubes (Büchi Electrothermal Mod.9100) and are uncorrected.Elemental analyses were performed on a Fisons elemental analyzer, Model EA1108CHN, and the data for C, H and N are within ± 0.4% of the theoretical values.

General Procedure for the C-7 Nucleophilic Substitution Reaction (Method A)
A mixture of the selected synthon (1.0 equiv), the appropriate base (3.0 equiv), and K 2 CO 3 (3.0equiv) in dry DMF was stirred at 40-80°C until no starting material was detected by TLC (2-72 h).After cooling, the reaction mixture was poured into ice/water yielding a precipitate which was washed with water and then with Et 2 O, and further purified as reported in the description of the compounds.

General Procedure for Reduction of C-6 Nitro Group (Method B)
A stirred solution of the selected 6-nitroderivative in DMF was hydrogenated over a catalytic amount of Raney nickel under atmospheric pressure at room temperature until no starting material was detected by TLC (15 min-3 h).The mixture was then filtered over Celite, and the filtrate was evaporated to dryness to afford a residue that was treated with EtOH/Et 2 O yielding a solid that was filtered and dried.

General Procedure for the Cycloaracylation Reaction, Step 1 (Method D)
A mixture of acrylate (1.0 equiv) and appropriate amine (1.2 equiv) in a mixture of Et 2 O/EtOH (3:1), was stirred at room temperature until no starting material was detected by TLC (30 min-2 h).The reaction mixture was then concentrated in vacuo, yielding a residue which was used in the consecutive step without further purification.

General Procedure For The Cycloaracylation Reaction, Step 2 (Method E)
A mixture of acrylate intermediate (1.0 equiv) and K 2 CO 3 (3.0equiv) in dry DMF was heated at 80°C for 1 h.After cooling, the reaction mixture was poured into ice/water, giving a precipitate that was filtered, washed with water and then with Et 2 O.

In vitro Anti-HIV Assays
The evaluation of the antiviral activity of the target compounds against HIV-1 strain III B in MT-4 cells was performed using the MTT assay as previously described [35,36].Mock-infected cells were used to assess the cytotoxic effects of the test compounds.

Fluorescence Quenching Assay (FQA)
The effect of tested quinolones on the Tat-TAR complex was evaluated using an FQA, a FRET-based competition assay, as previously described [23], using the Tat-derived peptide labelled with the donor fluorescein at its N terminus and the 29 nt wtTAR labeled at its 3'-end with a dabcyl moiety (quencher).

Chemistry
The introduction of the guanidine group at C-6 position in the quinolone derivative 1 was accomplished by reacting WM5 [20] with N, N′-di-(tert-butoxycarbonyl)thiourea in the presence of HgCl 2 followed by Boc deprotection of intermediate 13 under acid conditions Scheme (1).
The synthesis of the C-7 4-carbamimidoylpiperazine derivative 2 started by reacting derivative 14 [28] with methyl imidothiocarbamate sulfate to give intermediate 15, which, after reduction of the nitro group accomplished with FeSO 4 and NH 4 OH followed by acid hydrolysis of the ethyl ester intermediate, led to the target compound 2 Scheme (2).
Nitro derivative 19 was then catalytically reduced to amino derivative 20 and finally hydrolysed under basic conditions to give the target compound 3.The required building block 18 was in turn prepared through the reaction of nitrile derivative 16 [29] with ethylenediamine in presence of p-toluenesulfonic acid.
In the case of compound 26, the nucleophilic reaction led to the mixture of the di-substituted 29 and mono-substituted 30, which were easily isolated by chromatographical purification.Intermediate 30 was then functionalized at the N-1 position by reaction with piperazine in presence of DIPEA in dry DMF affording derivative 32.Then, the 6-nitro-derivatives 28, 29, 31, and 32 were catalytically reduced to give 6-amino-derivatives 33-36.Finally, the target compounds 4 and 6-8 were obtained by saponification of the corresponding ethyl esters 33-36.

Biological Evaluation
The newly synthesized compounds were initially evaluated for anti-HIV-1 (III B ) activity in MT-4 cells, determining their cytotoxicity in parallel Table 1.WM5 [20] was evaluated in the same cell lines for comparative purposes.
From the results, it emerged that the introduction of a C-6 guanidine group (compound 1) as well as an amidine moiety and a 4-imidazolinpyridine ring at the C-7 position (compounds 2 and 3) was completely detrimental in the inhibition of HIV replication.However, the low cytotoxicity showed by the compounds could suggest their inability to enter the cells.
The introduction of a 2-pyridinylmethyl moiety at N-1 position (compound 4) did not produce an active derivative (EC 50 > 19.8 µM).Conversely, the presence of a (pyridin-2-yl) piperazine spaced by an ethylene or propylene unit permitted to restore the anti-HIV-1 activity in compounds 5 and 6 (EC 50 = 5.05 and 1.21 µM, respectively) at no cytotoxic concentrations.
By alternatively deleting the piperazine or pyridine ring in compound 6, no activity at subcytotoxic concentrations was observed for compound 7, while only a weak inhibitory activity was conserved by compound 8.More interesting results were achieved when protonable side chains were inserted at the N-1 position of the 1,8-naphthyridone scaffold.In particular, the introduction of the (pyridin-2-yl)piperazine spaced by an ethylene or propylene unit gave compounds 11 and 12, both endowed with good anti-HIV-1 activity (EC 50 = 3.37 and ≥0.67 µM) coupled with SI values of 3.5 and ≤16, respectively.The deletion of the piperazine ring, as in compound 9 and 10, also in this case permitted to maintain anti-HIV-1 (EC 50 = 1.16 and ≥0.18 µM, SI = 3 and ≤18, respectively), with compound 10 that emerged as the most potent of the series.
In order to investigate the ability of active anti-HIV-1 compounds (derivative 5-7 and 9-12) to interfere with the formation of the Tat-TAR complex, they were evaluated in a FQA using a truncated Tat peptide.Unfortunately, solubility issues did not allow the evaluation of compounds 9, 11 and 12. Derivatives 5, 6, and 10 appeared to be able to inhibit the Tat-TAR complex formation, all presenting Ki values even improved as compared to the values obtained with WM5 Table 1.In particular, enhancing the distance of the pyridine ring from the quinolone nucleus, the ability of the compounds to inhibit the formation of the complex peptide-TAR increases: Ki 6 < 5 < 7; moreover, the presence of a piperazine in the side chain (compounds 6 and 5) has a positive effect on the activity of compounds, probably because it confers more rigidity to the chain and the position of the pyridine ring respect to the quinolone nucleus is fixed.Even if a strict correlation between the Ki values and the anti-HIV activities did not exist, this assay confirmed that the impairment of the Tat-TAR complex formation represents a putative target for the new derivatives.

CONCLUSION
The Tat-mediated transcription, the only phase of the HIV-1 replicative cycle in which viral genome amplification occurs, is a really attractive step that if inhibited by a suitable inhibitor could lead to a functional cure or even HIV infection eradication.For years, the inhibition of the Tat-mediated transcription has been highly pursued through different approaches, but this step of the viral replicative cycle remains untouched by any of the drugs in therapy.However, there are examples of promising Tat-mediated transcription inhibitors worthy to be mentioned, such as triptolide [37] and dCA [38] working on Tat protein.
In the attempt to obtain potent TAR binders, we started from WM5, a small molecule endowed with potent anti-HIV and Tat-mediated transcription inhibition activity thanks to the ability to selectively bind the bulge region of TAR; indeed no interaction with TAR-unrelated nucleic acids such as tRNA sequence and single-stranded or double-stranded DNA structures was observed [39].
Starting from WM5, various protonable moieties were inserted in different positions of the quinolone scaffold, intended to mime the basic region of Tat involved in the TAR interaction.The modifications introduced in the structure do not result in loss of activity, but rather seem to improve the activity as inhibitors of the Tat-TAR complex formation, leading to the identification of compounds able to recognize TAR better than WM5.Important SAR insights were also achieved for the quinolone class of anti-HIV derivatives.In particular, the C-6 and C-7 positions were confirmed as particularly sensitive to structural modifications, while the N-1 emerged as a suitable position to host protonable moieties such as the pyridine-based side chains.This is an important new insight since the most potent quinolones reported until now were all characterized by the presence of small alkyl groups at the N-1 position [22,40].In addition, potent anti-HIV compounds were identified, with naphthyridone derivatives 10 and 12 which showed EC 50 values in the sub-micromolar range.
In conclusion, the addition of protonable chains as tool to improve the TAR binding properties permitted to obtain active compounds, but additional knowledge on the interaction of TAR RNA with viral protein Tat and host Super Elongation Complex (SEC) [41,42] are still necessary to design compounds able to better recognize the TAR RNA and potently inhibit HIV replication.During the reviewing of this work, two interesting papers were published that could help the design of an effective TAR binder.In particular, Schulze-Gahmen and Hurley solved the crystal structure of the TAR in complex with Tat and the SEC core (CycT1/CDK9/AFF4) [43], while Varani and co-workers reported the NMR structure of TAR RNA in complex with a ultra-potent macrocyclic peptide that mimics the Arginine Rich Motif (ARM) of Tat [44], which however only weakly inhibit the Tat-mediated transcription.
These studies revealed how the direct interaction of Tat-ARM with TAR RNA bulge is required to induce the RNA structural change, but it is the recognition of the loop by the CycT1 that dominantly contributes to the binding energy.Thus, a future TAR binder should be able to target not only the UCU bulge but above all the interaction of CycT1 with the rearranged TAR RNA loop, to achieve an effective Tatmediated transcription inhibition in cells.

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