Synthesis of New 1,2,3-Triazol-4-yl-quinazoline Nucleoside and Acyclonucleoside Analogues

In this study, we describe the synthesis of 1,4-disustituted-1,2,3-triazolo-quinazoline ribonucleosides or acyclonucleosides by means of 1,3-dipolar cycloaddition between various O or N-alkylated propargyl-quinazoline and 1'-azido-2',3',5'-tri-O-benzoylribose or activated alkylating agents under microwave conditions. None of the compounds selected showed significant anti-HCV activity in vitro.


Introduction
An estimated 150 million people worldwide are chronically infected with hepatitis C virus (HCV) and have an increased risk of eventually developing liver cirrhosis or liver cancer [1]. We believe that a successful approach to cure HCV in most patients will likely require treatment with a combination of drugs that attacks different mechanisms necessary for replication and survival of HCV. Currently, patients undergo treatment with a combination of pegylated interferon alpha and ribavirin or a virusspecific protease inhibitor like telaprevir or boceprevir [2][3][4].
Heterocyclic structures are the basic elements of many pharmaceuticals, agrochemicals and veterinary products. Quinazolinone derivatives are an important class of these heterocyclic compounds that has been shown to display a broad-range of biological activities, for example, anticancer, diuretic, anti-inflammatory, anti-convulsant and antihypertensive activities [5][6][7].
In addition, 1,2,3-triazole nucleosides and carbanucleosides are N-Heterocyclic compounds which have been the subject of considerable research, mainly due to their value in synthetic organic chemistry [8][9][10][11][12][13][14] based on the Sharpless-Meldal modified Huisgen reaction. The classical 1,3-dipolar cycloaddition of azides and alkynes discovered by Huisgen [15] often gives mixtures of regioisomers (1,4-and 1,5-disubstituted triazoles). "Click Chemistry" is a term that was developed by Sharpless and independently by Meldal to illustrate a regioselective 1,3-dipolar cycloaddition using Cu(I) salts as catalyst. The catalyst can be added directly in the form of Cu(I) or Cu(II) salts using reducing agents to form active Cu(I) in-situ [16,17]. Cu(I) salts require at least an amine base to form the Cu-acetylide complexes. Many studies have shown that the presence of base under the process conditions provides stability for Cu(I) salts against oxidation. It has been used especially in anhydrous media and also under catalytic conditions [18].
In addition, the combination of two different and independently linked hybrid compounds can display synergy and result in a pharmacological potency greater than the sum of each individual moiety's potencies. For instance, nucleoside analogues incorporating triazole units are a valuable area of therapeutic research, and some triazole-containing compounds have shown activities against hepatitis and HIV-1 [19,20].
The poor treatment response, combined with often-severe side effects induced by therapy, highlights the need for improved antiviral drugs with better efficacy and safety profiles. Furthermore, in continuation of our research program centered on click chemistry [28,29], the aim of the present work was to synthesize some new hybrid compounds combining the two heterocycles: quinazolinone and 1,2,3-triazole. The new compounds were also assessed for their anti-HCV activities.

Synthesis of Protected Nucleosides and Acyclonucleosides 6a-i
Typically, quinazolinone structures were constructed using anthranilic acid or its derivatives via a sequence of acylation and condensation, which requires strong acidic or basic reaction conditions [30][31][32]. The quinazoline derivatives were prepared from anthranilic acid (1) in three steps. Initially, the acid was reacted with benzoyl chloride in anhydrous pyridine at 0-5 °C for one hour. Afterwards the reaction mixture was stirred (two hours) at room temperature until 2-phenylbenzoxazinone (2a) was formed [33,34]. Alternatively, 2-methylbenzoxazinone (2b) was obtained by reaction of anthranilic acid with acetic anhydride using microwave irradiation [35,36]. The benzoxazinones were further treated with formamide under microwave irradiation to obtain the quinazolinones 3a-b. On the other hand, the synthesis of quinazolin-4-one 3c was achieved by condensing anthranilic acid with 2.5 equivalents of formamide under microwave irradiation [37] (Scheme 1).  The alkylation of quinazol-4-ones which are substituted in position 2 sometimes leads to two isomers resulting from competing N-alkylation and O-alkylation and the ratio of these isomers depends on the substituent at position 2. An earlier study confirmed that the substituents and the reaction conditions play a significant role in influencing the ratio of O-alkylation vs. N-alkylation [38]. In this investigation, the quinazolinones 3a-c were treated with propargyl bromide in the presence of potassium t-butoxide. The reaction was carried out using DMF as the solvent [39][40][41]   The propargylated quinazolines 4 and 5 were used in the 1,3-dipolar cycloaddition reaction to link 1,2,3-triazole with the quinazoline nucleus to yield compounds 6. Reaction of the triple bonds of propargylated quinazoline and the azide of sugar and pseudo-sugar was performed under microwave irradiation using Cul as catalyst without solvent (Scheme 3). This protocol (click chemistry) for the formation of the triazole rings is efficient, easy and convenient and typically gives almost quantitative reaction yields [42]. The cycloaddition reaction is drawn in Scheme 3 and the obtained products are tabulated in Table 1. We characterized the structures of all products by 1 H-NMR, 13 C-NMR and mass spectrometry. In addition, the structures of 6g and 6i were confirmed by X-ray crystallographic analysis. The structure of crystal (a) illustrates that the anomeric configuration at the C1'-stereocentre has not been affected during the click reaction, the steric effect of benzoyl group in the position 2' and 3' (ribose) directs the reaction towards the β-configuration. The crystal structures of these compounds are shown in Figure 1 [43,44].

Deprotection of Nucleosides and Acyclonucleosides: Preparation of 7a-i
For deprotection base catalyzed methods were employed. Sodium methoxide (NaOMe) in methanol was used for the deprotection of the benzoyl group of compounds 6a,d,g [45]. On the other hand, the deprotection of the acetyl group of compounds 6b,c,e,f,h,i was carried out using potassium carbonate (K 2 CO 3 ) in methanol (Scheme 3 and Table 1). The structure of 7c was confirmed by X-ray crystallographic analysis. The crystal structure of this compound is shown in Figure 2 [46]. Finally, we were also interested in studying the biological activity of 1,2,3-triazole ribonucleosides 7a-i. These derivatives were tested in vitro to evaluate their anti-HCV activity. None of the new compounds were found to inhibit HCV replication in vitro ( Table 2). Antiviral activity was assessed in a 3-day cell culture assay using the HCV-replicon-containing cell line, AVA5 (genotype 1b, CON1) (provided to GUMC by Apath, Inc., Brooklyn, NY, USA) as previously described [47].

General
1 H-and 13 C-NMR spectra were recorded in CDCl 3 or DMSO-d 6 on a Bruker 300 MHz instrument using SiMe 4 as internal standard. Chemical shifts are given in ppm and coupling constants (J) in MHz (br, broad; m, multiplet; t, triplet; d, doublet; and s, singlet). Mass spectra were obtained using ESI/MS and MALDI-TOF-MS. Reactions were carried out in a microwave oven Model AVM510/WP/WH. The reactions were controlled by thin layer chromatography (TLC) on precoated silica gel 60 F254 (Merck, Darmstadt, Germany); UV light was used for visualization of the spots. All products were purified by column chromatography on silica gel (100-200 mesh) Merck. [36] Initially, anthranilic acid (1, 2 g, 14.5 mmol) was dissolved in dry pyridine (20 mL). Then benzoyl chloride (1.1 equiv) was added dropwise at 0 °C. The reaction mixture was maintained at 0 °C for 1 h and then allowed to stir at room temperature for 2 h, during which time a solid product precipitated out. The mixture was neutralized using a saturated solution of sodium bicarbonate.

Synthesis of Propargylated Quinazolines
The appropriate quinazolin-4-one 3a-c (2 mmol) was dissolved in dry DMF (2.5 mL); KOt-Bu (1.1 equiv) was added. The mixture was stirred for 15 min at room temperature. Afterwards, propargyl bromide (2.5 mmol) was added dropwise to the mixture. The reaction was performed for 15 min at room temperature. The reaction mixture was diluted with water (10 mL) and extracted with ethyl acetate (2 × 20 mL); the organic phase was dried over Na 2 SO 4 and evaporated under vacuum. The crude products were purified by column chromatography using CH 2 Cl 2 /MeOH = 99:1, v/v as eluent.

Benzoyl Group Deprotection
The compound (6a,d,g, 1 mmol) was dissolved in dry methanol (2.5 mL). NaOMe (1 eq) was added to the solution with stirring for 30 min at room temperature. The neutralization was performed with AmberliteIR120 hydrogen form. After, filtration and evaporation the residue was purified by silica gel flash column chromatography.

Acetyl Group Deprotection
A solution of (6b,c,e,f,h,i, 1 mmol) in dry methanol (2.5 mL) was treated with 1 eq of K 2 CO 3 . The reaction mixture was stirred at room temperature for 15 min. The deprotected compound was purified by silica gel flash column chromatography.

Conclusions
In summary, the synthesis of a series of 1,2,3-triazole-4-yl-quinazoline derivatives starting from anthranilic acid was performed efficiently using click chemistry under microwave irradiation. None of the compounds selected showed significant anti-HCV activity.