Synthesis , Cytostatic and Antibacterial Evaluations of Novel 1 , 2 , 3-Triazolyl-tagged Pyrimidine and Furo [ 2 , 3-d ] pyrimidine Derivatives

C-5 alkynylated and N-1 alkylated pyrimidine derivatives were synthesized by N-alkylation reaction of 5-iodouracil in the presence of NaH, as a base, followed by Pd-catalyzed Sonogashira cross-coupling reaction of N-alkyl-5-iodouracil derivatives (1 and 2) with corresponding terminal alkynes. Intramolecular in situ O-heteroannulation ring closure of N-1-alkyl-C-5-alkynylpyrimidine derivatives (3 and 5) generated novel 6-substituted furo[2,3-d]pyrimidine derivatives (7 and 8). 1,4-Disubstituted 1,2,3-triazole tethered 5-alkynylpyrimidines (14–19) and 6-substituted furo[2,3-d]pyrimidines (20–22) were successfully prepared by the copper(I)-catalyzed click reaction of 5-iodo-N-1-propargylpyrimidine (2) using microwave irradiation, followed by Sonogashira cross-coupling reaction with corresponding terminal alkynes. In vitro antiproliferative activity of prepared compounds evaluated on human cancer cell lines cervix adenocarcinoma (HeLa), colon adenocarcinoma (CaCo-2), chronic myeloid leukemia in blast crisis (K562), Burkitt lymphoma (Raji) revealed that pyrimidine (19) and furo[2,3-d]pyrimidine (22) derivatives with 3,5-difluorophenyl at pyrimidine and furo[2,3-d]pyrimidine as well as p-(trifluoromethyl)phenyl at 1,2,3-triazole exhibited marked and selective inhibitory effects on the growth of K562 and Raji tumor cells. Antibacterial evaluations showed that pyrimidine derivative 14 substituted with p-tolylethynyl at C-5 of pyrimidine and benzyl at 1,2,3-triazole moiety was the most active of all evaluated compounds on the Gram positive bacterial strains Enterococcus faecalis. Further structure optimization of compounds 14, 19 and 22 is foreseen in order to obtain lead structural analogs with efficient and selective antitumoral and antibacterial activities.


Antibacterial Evaluations
The in vitro antibacterial activity of novel pyrimidine and furo [2,3-d]pyrimidine derivatives was tested against Gram-positive bacteria including Staphylococcus aureus (ATCC 25923), Enterococcus faecalis, vancomycin-resistant Enterococcus faecium (VRE) and Gram-negative bacteria including Pseudomonas aeurigonsa (ATCC 27853), Escherichia coli (ATCC 25925), Acinetobacter baumannii (ATCC 19606), extended-spectrum -lactamase (ESBL)-producing Klebisiella pneumoniae (Table 2).The obtained results were compared with known antibiotic ciprofloxacin (CIP).As displayed in the Table 2, almost all tested compounds did not show antibacterial activities on the growth of evaluated Gram-positive and Gramnegative bacterial strains, except for pyrimidine derivative 14 with p-tolylethynyl substituent at C-5 and benzyl at 1,2,3-triazole which is the most active of all evaluated compounds on the Gram positive bacterial strains Enterococcus faecalis (MIC = 8 µg/mL).

Materials and General Methods
Commercially available chemicals were purchased from Sigma Aldrich (Germany) and Acros (Belgium) and where used without purification.All solvents used in synthesis were analytical grade purity and dried.Dichloromethane (CH2Cl2) was stored over 4 Å molecular sieves.Methanol (CH3OH) and tert-buthanol (t-BuOH) were stored over 3 Å molecular sieves without distillation.Melting points were determined on a Kofler micro hot-stage instrument (Reichter, Wien) and were uncorrected.Precoated Merck silica gel 60 F254 plates were used for thin-layer chromatography and spots were visualized by shortwave UV light (254 nm).Column chromatography was performed on Fluka silica gel (0.063-0.200 mm), with dichloromethane : methanol and dichloromethane as mobile phases.Microwave-assisted syntheses were performed in a Milestone start S microwave oven using glass cuvettes at 80 °C and 300W under the pressure of 1 bar.NMR spectroscopy 1 H and 13 C NMR spectra were recorded on a Varian Gemini 300 spectrometer (Ruđer Bošković Institute, Zagreb).Samples were measured in DMSO-d6 solutions at 25 °C in 5 mm NMR tubes. 1 H and 13 C NMR chemical shifts (δ) in ppm were referred to TMS (δ 0.0 ppm).Individual resonances were assigned on the basis of their chemical shifts, signal intensities, multiplicity of resonances and H-H coupling constants.The electron impact mass spectra and the purity of compounds were assessed by using Agilent Technologies 6410 Triple Quad LC/MS instrument equipped with electrospray interface and triplequadrupole analyzer (LC-MS/MS) in positive instrument mode.High performance LC was performed on Agilent 1100 series systemwith UV detection (photodiode array detector) using Zorbax C18 reverse-phase analytical column (2.1-30 mm, 3.5 mm).All compounds used for biological evaluation showed >95 % purity in HPLC-MS/MS system.

Biological Evaluations
Cell Cultured Cells were cultured in tissue culture flasks (25, 75 cm 2 ) in humidified atmosphere under the conditions of 37 °C / 5 % of CO2 gas in the CO2 incubator (IGO 150 CELLlife TM , JOUAN, Thermo Fisher Scientific, Waltham, MA, USA).HeLa, CaCo-2 and MDCK I were maintained in DMEM medium complemented with 10 % heat-inactivated FBS, 2 mM glutamine, and 100U / 0.1 mg penicillin / streptomycin.Cell lines in suspension, K562 and Raji, were cultured in RPMI-1640 medium complemented with 10 % heat-inactivated FBS, 2 mM glutamine, 1 mM Na-pyruvate, and 10 mM HEPES.Cell viability was assessed by the trypan blue dye exclusion method before each experiment.

Cytotoxicity Evaluation
Cytotoxic effects on the tumors cell growth were determined using the colorimetric methyltetrazolium (MTT) assay.Experiments were carried out on four tumor human cell lines (HeLa, CaCo-2, K562 and Raji) and on one canine cell line (MDCK I) as normal cells.The adherent cells were seeded in 96 micro-well plates at a concentration of 2 × 10 4 cells/mL and allowed to attach wall plate overnight in the CO2 incubator.After 72 hours of incubation with tested compounds, the medium was replaced with 5 mg/mL MTT solution and the resulting formazane crystals were dissolved in DMSO.Suspension cells (K562 and Raji) at a concentration of 1 × 10 5 cells/mL, were plated onto 96 microwell plates and the same day were treated with tested extracts at different concentrations.After expired 72 hours of incubation, 5mg/mL MTT solution was added to each well and incubated 4 hours in CO2 incubator.To each well, 10 % SDS with 0.01 mol/L HCl was added to dissolve water-insoluble MTT-formazane crystals overnight.Elisa micro plate reader (iMark, BIO RAD, Hercules, CA, USA) was used for measurement of absorbance at 595 nm.
All experiments were performed at least three times in triplicates.The percentage of cell growth (PG) was calculated using the following equation

Table 1 .
Inhibitory effects of pyrimidine and furo[2,3-d]pyrimidine derivatives on the growth of human tumor cell lines HeLa, CaCo-2, Raji and K562 and normal Madin Darby canine kidney (MDCK I) cells as well.