Synthesis of 1,4-Disubstituted Mono and Bis-triazolocarbo-acyclonucleoside Analogues of 9-(4-Hydroxybutyl)guanine by Cu(I)-Catalyzed Click Azide-Alkyne Cycloaddition

A series of novel mono-1,2,3-triazole and bis-1,2,3-triazole acyclonucleoside analogues of 9-(4-hydroxybutyl)guanine was prepared via copper(I)-catalyzed 1,3-dipolar cycloaddition of N-9 propargylpurine, N-1-propargylpyrimidines/as-triazine with the azido-pseudo-sugar 4-azidobutylacetate under solvent-free microwave conditions, followed by treatment with K2CO3/MeOH, or NH3/MeOH. All compounds studied in this work were screened for their antiviral activities [against human rhinovirus (HRV) and hepatitis C virus (HCV)] and antibacterial activities against a series of Gram positive and negative bacteria.


Introduction
For several years, there has been an intensive search for drugs effective in chemotherapy of viral diseases like AIDS, herpes simplex, Hepatitis C and cytomegaloviruses [1][2][3][4][5]. Most of these drugs are analogues of naturally occurring nucleosides [6]. A series of nucleoside analogues were synthesised in which the cyclic carbohydrate moiety was replaced by an acyclic side chain [7][8][9][10][11][12]. The biological OPEN ACCESS activities of acyclonucleosides, after the discovery of acyclovir [9-((2-hydroxyethoxy) methyl)guanine ACV (Zovirax)] (1, Figure 1), have led to the synthesis of a diversity of structures. Many variations were tested in order to enhance biological activity and selectivity, or to lower toxicity [13][14][15][16][17][18]. Among them HBG [9-(4-hydroxybutyl)guanine] (2, Figure 1) presented good activity against HSV-1 and HSV-2. (1) (2) (3) O NH 2 On the other hand, for antiviral agents, triazolonucleosides and acyclonucleosides have attracted much attention. Ribavirin (3, Figure 1), whose nucleobase consists of an unnatural triazole moiety, was the first synthetic nucleoside to show a broad spectrum of antiviral activities against many RNA and DNA viruses [19]. Furthermore, nucleosides with unnatural triazole nucleobases are generally resistant to nucleos(t)ide metabolizing enzymes, and this may lead to better in vivo stability and efficiency. Because of their broad application as pharmaceuticals like antibacterial or antiviral agents, a great number of 1,2,3-triazole derivatives have been reported as potent antiviral, antimicrobial or antiproliferative agents [20]. Also the synthesis and biological evaluation of carbonucleosides (substances in which the anomeric oxygen of the furanose ring is replaced by a methylene group), having a 1,2,3-triazole ring as a nucleobase (e.g., 4, Figure 1) have been reported. Until now, very few efforts were made on appending aromatic systems to triazole nucleosides. We expect that these extended aromatic systems may offer advantageous binding properties to the corresponding biological targets via larger aromatic systems.

Results and Discussion
Different synthetic methods have been developed for the construction of triazole frameworks. These compounds are typically prepared by thermal cycloaddition of azides and alkynes [21,22]. Two problems are, however, encountered in this transformation: (1) reactivity of the substrates, either alkynes or azides require activation by an electron withdrawing group, otherwise, the reaction must be carried out at higher temperatures; (2) the regioselectivity of the products, as for unsymmetrical alkynes, a mixture of regioisomers is obtained in most cases. Since Sharpless reported copper(I) catalysis for regioselective cycloaddition of terminal alkynes and azides to yield exclusively 1,4-disubstituted-1,2,3-triazoles, many groups have reported their results employing different kinds of Cu(I) salts as catalyst [23][24][25][26][27][28][29][30][31][32][33][34][35]. In addition, microwave irradiation has become a powerful synthetic tool for rapid synthesis of a variety of biologically active compounds. Its use to is to enhance the rates of classical organic reactions.
In the light of these findings and in continuation of our previous investigation [34], we considered the synthesis of new 1,2,3-triazole and bis-1,2,3-triazole acyclonucleosides. They carry either a purine, pyrimidine or as-triazine moiety as nucleobase appended to 1,2,3-triazole. They can be regarded as analogues of 9-(4-hydroxybutyl)guanine (HBG). We went further to combine nucleobase and triazole rings with an acyclic side-chain developed bistriazolyl acyclonucleosides, and determined their in vitro antiviral and antibacterial activities.

Chemistry
The starting material 4-azidobutylacetate (7)  The second step of the synthesis was the preparation of monopropargylated nucleobases. For this, uracil, thymine, 6-azauracil and adenine were used as starting materials that were treated with propargylbromide in the presence of K 2 CO 3 . All reactions were carried out in DMF, as it is an excellent solvent for dissolving nucleobases [34] (Scheme 2). The pyrimidine and as-triazine derivatives were exclusively alkylated at the N-1 position, (9a-c), and the purine in N-9 position, (9d) as confirmed by 1 H-NMR and 13 C-NMR spectra.

Scheme 2.
Preparation of mono-triazolo-carboacyclonucleosides 10a-d. The terminal triple bonds of propargylated nucleobases were ligated to the azide residue of the pseudosugar using copper(I)-catalyzed 1,3-dipolar cycloaddition and Et 3 N under microwave-assisted reaction without solvent [34] (Scheme 2) leading to the 1,4-disubstituted regioisomer in a quantitative yield unlike before [22] and a reaction time of one minute (Table 1). We intimately mixed the azide, acetylenic derivative and copper(I)-iodide prior to microwave irradiation. This fast and efficient method was in all tested cases superior in yield and handling to running the reaction in solution [34]. Table 1. Structures of the starting azides, alkynes and corresponding products.   A common feature of many acyclic nucleoside analogues showing biological activity, including HBG, is the presence of a primary alcoholic group. This function and the nucleic acid base are essential for their biological activity. For this purpose the deprotected products were obtained in good yields by treatment with NH 3 /methanol or K 2 CO 3 /methanol.

Entry
To extend the general applicability of the microwave assisted click reaction for the synthesis of triazole acyclonucleosides we included other alkinyl derivatives, as outlined in Scheme 2. Analogously to the preparation of N-1-propargylated pyrimidines/as-triazine, the N-1, N-3-bis-propargylated pyrimidines/as-triazines were prepared from N-1-propargylated uracil, thymine and 6-azauracil (Scheme 3), (Yields 80-85%). The bis-propargylated pyrimidines/as-triazines were converted into the bis-triazole acyclonucleosides using the same reaction conditions in an almost quantitative yield (Table 1).

Antibacterial Activity
The  [37]. Ciprofloxacin and linezolid were used as standard drugs for comparison ( Table 2). As shown in Table 2, no antibacterial activities against Gram-positive and Gram-negative bacteria were noted. All compounds showed antibacterial activity with a range of the MICs higher than 64 µg/mL.

Antiviral Activity and Cytotoxicity
Antiviral activities of the synthesized compounds were screened against two types of viruses in human epithelial (HeLa) cells for HRV and Human hepatocarcinoma (Huh) cells for HCV. For each compound, the 50% and 90% effective concentration (EC 50 , EC 90 ) and the minimal toxic concentration (MTC) or the 50% cytotoxic concentration (CC50) was obtained. None of the compounds exhibited specific antiviral activity, which means that they did not inhibit the replication (induction of viral cytopathogenicity) of any of the viruses tested.

General Procedure for the Synthesis of Monopropargyl Heterocyclic Bases
The mixture of heterocyclic base (thymine, uracil, 6-azauracil and adenine, 1 mmol), K 2 CO 3 (0.5 mmol) and propargyl bromide (1 mmol) in anhydrous DMF (20 mL) was stirred at room temperature during 24 h. After removal of the solvent under reduced pressure the residue obtained was purified on a silica gel column eluted with CH 2 Cl 2 and MeOH (99/1).

General Procedure for the Synthesis of the N-1, N-3-Bis-propargylpyrimidines/as-Triazines
The mixture of the heterocyclic base (N-1-propargyluracil, N-1-propargylthymine, and N-1propargyl-6-azauracil, 1 mmol), K 2 CO 3 (0.5 mmol) and propargylbromide (1.1 mmol) in anhydrous DMF (20 mL) was stirred at room temperature during 15 h. After removal of the solvent under reduced pressure and purification on silica gel column chromatography, we obtained the desired pure product.

General Procedure for the Synthesis of the Triazole acyclonucleoside Derivatives
The mixture of alkylazide (5 mmol), Et 3 N (1 mmol), N-propargylbase (1 mmol) and CuI (0.1 mmol) was irradiated in the microwave oven at power level (300 W) for 1 min without solvent. K 2 CO 3 (2 mmol) in methanol (10 mL) was added directly to reaction mixture. The mixture was stirred for additional 3 h at room temperature (or in 30 mL of methanol saturated with ammonia at 0 °C during 24 h). When TLC analysis showed no starting material, solvent was removed under reduced pressure, and the residue was purified on silica gel eluting with dichloromethane and methanol.

Conclusions
A series of triazole carboacyclonucleosides with various nucleobase moieties appended on the triazole were synthesized efficiently using a convenient one-step click azide-alkyne cycloaddition reaction under solvent-free microwave irradiation. All compounds synthesized were evaluated for their antibacterial and antiviral activities but none exhibited specific activity so far. Further applications of the click azide-alkyne cycloaddition process are currently under investigation and will be reported in due course.