Phosphonylated Acyclic Guanosine Analogues with the 1,2,3-Triazole Linker

A novel series of {4-[(2-amino-6-chloro-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}alkylphosphonates and {4-[(2-amino-6-oxo-1,6-dihydro-9H-purin-9-yl)methyl]-1H-1,2,3-triazol-1-yl}alkylphosphonates as acyclic analogues of guanosine were synthesized and assessed for antiviral activity against a broad range of DNA and RNA viruses and for their cytostatic activity toward three cancerous cell lines (HeLa, L1210 and CEM). They were devoid of antiviral activity; however, several phosphonates were found slightly cytostatic against HeLa cells at an IC50 in the 80–210 µM range. Compounds (1R,2S)-17k and (1S,2S)-17k showed the highest inhibitory effects (IC50 = 15–30 µM) against the proliferation of murine leukemia (L1210) and human T-lymphocyte (CEM) cell lines.

Generally, in order to secure sufficient bioavailability of active phosphonate nucleotide analogues, they are administered as prodrugs, namely the respective phosphonate esters or amides [33][34][35][48][49][50][51][52][53]. For this reason, we designed guanosine analogues 16 and 17 as the respective phosphonate esters to ensure sufficient membrane permeability. Moreover, our recent experiences clearly supported the strategy to prepare diesters rather than free phosphonic acids, which are completely ionized at physiological pH. Indeed, we found several examples of active diesters in the class of 1,2,3-triazole phosphonates, whereas the respective free acids appeared inactive [38,39,54].
Although cycloadditions of propargylated guanines 18a (R = H) and 18b (R = Ac) to various azides have previously been mentioned [41][42][43], in our hands, the reaction of 18a, as well as 18b with the azidomethylphosphonate 14a failed because of the low solubility of guanines 18a and 18b (Scheme 2). Attempts at running a cycloaddition of the phosphonate 14a with propargylguanine 18a at 110 °C in toluene resulted in the recovery of starting materials only. Similarly, when the 3-azidopropylphosphonate 14c was treated with 18a or 18b at 110 °C in toluene, as well as under microwave (MW) irradiation in aqueous ethanol, no traces of cycloadducts were observed. For this reason, we turned to 2-amino-6-chloro-9-propargylpurine 19, which was found to be sufficiently soluble in the reaction medium and reacted with the respective ω-azidoalkylphosphonates 14 to form the intermediate 2-amino-6-chloropurines 17, which were transformed into guanine analogues in the last step. Thus, azides 14 were subjected to cycloaddition with Compound 19 in the presence of Cu(I) salt under microwave irradiation to give 1,2,3-triazoles 17a-k. Reactions were complete at 35-40 °C within 15 min (Scheme 3). Subsequently, 17a-k were treated with 75% trifluoroacetic acid to provide acyclic guanosine analogues 16a-k in good yields (92%-98%). However, attempts at preparing (1R,2S)-16k and (1S,2S)-16k failed, since the treatment of (1R,2S)-17k and (1S,2S)-17k with trifluoroacetic acid led to severe decomposition. All final compounds were purified by chromatography, and solids were finally recrystallized; their purity was ascertained by NMR spectroscopic methods and elemental analysis.
The cytotoxicity of the tested compounds toward the uninfected host cells was defined as the minimum cytotoxic concentration (MCC) that causes a microscopically-detectable alteration of normal cell morphology. The 50% cytotoxic concentration (CC50), causing a 50% decrease in cell viability, was determined using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4sulfophenyl)-2H-tetrazolium (MTS) assay system. The cytostatic activity of the tested compounds was defined as the 50% cytostatic inhibitory concentration (IC50), causing a 50% decrease in cell proliferation, and was determined against murine leukemia L1210, human T-lymphocyte CEM and human cervix carcinoma HeLa cells (Table 1). Table 1. Inhibitory effect of the tested compounds against the proliferation of murine leukemia (L1210), human T-lymphocyte (CEM) and human cervix carcinoma cells (HeLa).
None of the tested compounds affected cell morphology of HEL, HeLa, Vero, MDCK and CRFK cells at concentrations up to 100 μM. Instead, several compounds appeared slightly cytostatic, selectively against HeLa cells at an IC50 in the 80-210 µM range. From the entire library of compounds, only (1R,2S)-17k and (1S,2S)-17k appeared to be the most active toward all tested cancerous cell lines, and they showed the highest inhibitory effect (IC50 = 16-30 µM) against the proliferation of murine leukemia (L1210) and human T-lymphocytes (CEM).
The significantly higher activity of dibenzyl phosphonates (1R,2S)-17k and (1S,2S)-17k is probably due to their better penetration through cell membranes when compared to the other compounds in the series 17, as well as 16, which all were tested as diethyl esters.

General
1 H-NMR were taken in CDCl3 or CD3OD on the following spectrometers: Varian Mercury-300 (Varian NMR Instrument, Palo Alto, CA, USA) with TMS as an internal standard; chemical shifts δ in ppm with respect to TMS; coupling constants J in Hz. 13 C-NMR spectra were recorded on Varian Mercury-300 (Varian NMR Instrument, Palo Alto, CA, USA) and Bruker Avance III spectrometers (Bruker Instruments, Karlsruhe, Germany) at 75.5 and 151 MHz, respectively. 31 P-NMR spectra were taken in CDCl3 or CD3OD on a Varian Mercury-300 (Varian NMR Instrument, Palo Alto, CA, USA) at 121.5 MHz.
IR spectral data were measured on an Infinity MI-60 FT-IR spectrometer (ATI Instruments North America-Mattson, Madison, WI, USA). Melting points were determined on a Boetius apparatus (VEB Kombinat NAGEMA, Dresden, DDR-Currently Germany) and are uncorrected. Elemental analyses were performed by the Microanalytical Laboratory of this faculty on a Perkin Elmer PE 2400 CHNS analyzer (Perkin-Elmer Corp., Norwalk, CT, USA).
The following adsorbents were used: column chromatography, Merck silica gel 60 (70-230 mesh); analytical TLC, Merck TLC plastic sheets silica gel 60 F254. TLC plates were developed in chloroform−methanol solvent systems. Visualization of spots was effected with iodine vapors. All solvents were purified by methods described in the literature.

General Procedure for Transformation 17 into 16
The respective 2-amino-6-chloropurine derivative 17a-17j (1.00 mmol) was dissolved in a 75% aqueous solution of trifluoroacetic acid (6 mL) and left at room temperature overnight. The solvent was removed, and the residue was co-evaporated with water and subsequently with ethanol to give pure guanine derivatives 16a-16j.

Antiviral Activity Assays
The compounds were evaluated against the following viruses: herpes simplex virus type 1 (HSV-1) strain KOS, thymidine kinase-deficient (TK − ) HSV-1 KOS strain resistant to acyclovir (ACV r ), herpes simplex virus type 2 (HSV-2) strains Lyons and G, varicella-zoster virus (VZV) strain Oka, TK − VZV strain 07−1, human cytomegalovirus (HCMV) strains AD-169 and Davis, vaccinia virus Lederle strain, respiratory syncytial virus (RSV) strain Long, vesicular stomatitis virus (VSV), Coxsackie B4, para-influenza 3, influenza virus A (subtypes H1N1, H3N2), influenza virus B, reovirus-1, Sindbis, reovirus-1, Punta Toro, human immunodeficiency virus type 1 strain IIIB and human immunodeficiency virus type 2 strain ROD. The antiviral, other than anti-HIV, assays were based on the inhibition of the virus-induced cytopathicity or plaque formation in human embryonic lung (HEL) fibroblasts, African green monkey cells (Vero), human epithelial cells (HeLa) or Madin-Darby canine kidney cells (MDCK). Confluent cell cultures in microtiter 96-well plates were inoculated with 100 CCID50 of virus (1 CCID50 being the virus dose to infect 50% of the cell cultures) or with 20 plaque forming units (PFU) (VZV) in the presence of varying concentrations of the test compounds. Viral cytopathicity or plaque formation was recorded as soon as it reached completion in the control virus-infected cell cultures that were not treated with the test compounds. Antiviral activity was expressed as the EC50 or compound concentration required to reduce virus-induced cytopathogenicity or viral plaque formation by 50%.

Cytostatic Activity Assays
All assays were performed in 96-well microtiter plates. To each well were added (5−7.5) × 10 4 tumor cells and a given amount of the test compound. The cells were allowed to proliferate for 48 h (murine leukemia L1210 cells) or 72 h (human lymphocytic CEM and human cervix carcinoma HeLa cells) at 37 °C in a humidified CO2-controlled atmosphere. At the end of the incubation period, the cells were counted in a Coulter counter. The IC50 (50% inhibitory concentration) was defined as the concentration of the compound that inhibited cell proliferation by 50%.