Synthesis, Biological and Anti-tumor Evaluation of Some New Nucleosides Incorporating Heterocyclic Moieties

1,3-diaryl-1-propen-3-ones 1a-h, were used as building blocks for a large range of nucleoside analogs incorporating five and six-membered heterocyclic rings. Heterocyclic compounds incorporating aromatic moieties (2-11) and their N-nucleoside analogs (13-20) were synthesized. New compounds were evaluated for their potential antimicrobial, antifungal activities and for their in vitro cytotoxic activity against three cell lines: human breast cancer cell line (MCF7), colon carcinoma cells (HCT) and human epidermid/arynx carcinoma cell line (HEp2).

The aim of this work was to design, synthesis of indazole, pyridines, 2-aminooxazines and/or 2-thiopyrimidine derivatives bearing aromatic moieties and use these compounds as a basis for the synthesis of a series of nucleosides. Some of the new compounds were then examined for cytotoxic activity via assays on human breast cancer cell line MCF-7, colon carcinoma cells (HCT), human epidermid/arynx carcinoma cell line (HEp2).

Experimental
All melting points for the prepared derivatives were measured in capillary tubes using a Gallen-Kamp apparatus and were uncorrected. The IR spectra were recorded on a Perkin-Elmer 1650 spectrophotometer (KBr pellets) and the wave numbers were given in cm-1. The 1H, 13C NMR spectra were measured in dimethyl sulphoxide-d6 as a solvent using a Varian Gemini 180 spectrometer operating at 300 MHz for 1H, and 75 MHz for 13C. TMS was used as an internal standard and the chemical shifts were reported as δ ppm. The FAB mass spectra were recorded on a JEOL SX 102/DA-6000 mass spectrometer.

Synthesis of 2-amino-4,6-diaryl-2H-1,3-oxazine derivatives (11a-c)
A mixture of 1d, f, h (0.01 mol) and urea (0.01 mol) in 15 ml of absolute ethanol containing 6 ml of glacial acetic acid was heated under reflux for 6 h. The precipitate that formed after cooling was collected, washed well with dilute ethanol and recrystallized from the proper solvent to give 11a-c, respectively.

Synthesis of nucleoside derivatives (13)
A suspension of 2 (0.01 mol) in 6 ml of aqueous potassium hydroxide (prepared by dissolving 0.01 mol in 6 ml of distilled water) was stirred by using a magnetic stirrer for 3 h, then a solution of 12 (0.0 mol) dissolved in 30 ml of dry acetone was added drop-wise while stirring which continued for 12 h. After evaporation of the solvent (reduced pressure), the residue was washed with dilute ethanol several times and the precipitate formed was recrystallised from ethanol to give 13 as brown crystals in 60% yield, m.p.

Synthesis of nucleoside derivatives 15 and 16
A suspension of the cyanopyridone derivatives 5c and/or 2-iminocyanopyridine 6c (0.01 mol) in 6 ml of aqueous KOH solution (prepared from dissolving (0.01 mol) solid KOH in 6 ml of distilled water) was well stirred (magnetic stirrer) at room temperature for 3 h, then a solution of 12 (0.01 mol) dissolved in 30 ml of dry acetone was added drop-wise while stirring. Stirring was continued for further 12 h. After evaporation of the excess solvent (reduced pressure), the residue left was washed with dilute alcohol (several times) and the precipitate formed was recrystallized from ethanol to give 15 and 16.

Synthesis of nucleoside derivatives (19a-c)
A suspension of pyrimidin-2-thione derivatives (10b,d,e) (0.01 mol) in 6 ml of aqueous KOH solution (prepared from dissolving (0.01 mol) solid KOH in 6 ml of distilled water) was well stirred (magnetic stirrer) at room temperature for 3 h, then a solution of 12 (0.01 mol) dissolved in 30 ml of dry acetone was added drop-wise while stirring. Stirring was continued for further 12 h. After evaporation of the excess solvent (reduced pressure), the residue left was washed with dilute alcohol (several times) and the precipitate formed was recrystallized from ethanol to give (19a-c).

Results and Discussion
1,3-diaryl-1-propen-3-ones, 1a-h, which were synthesized according to the literature [34], were used as starting material for the synthesis of a large range of heterocyclic compounds (compound series 2-6) as depicted in Scheme 1.
Reaction of compound 2 with 2,3,4,6-tetra-O-acetyl-α-Dglucopyranosyl bromide 12 gave the corresponding 13. Its infrared spectrum revealed the presence of absorption bands at 1663, 1600, and 3346 cm -1 due to CONH and NH/OH respectively. The 1 H-NMR (DMSO-d 6 ) of 13 showed a doublet for anomeric protons at δ 6.9, 7.0 ppm due to diaxial orientations of H-1' and H-2' protons, indicating their presence in the β-configuration and the other protons of the xylopyranosyl resonating in the region 3.3-3.7 ppm, while the protons of the two acetyl moieties showed as two singlets at δ 2.41 and 2.43 ppm. The presence of the two OH protons was indicated by two singlets at δ 10.04 and 10.52 ppm. The mass spectrum of 13 showed a molecular ion peak at m/z 575 indicating the partial hydrolysis of one acetyl and the metonym groups in the molecule to the corresponding OH group.
Similarly α-D-xylopyranosyl bromide 12 was reacted with the indazolone 4 under the same conditions to give the corresponding (2R,3S,4R,5S)-5-hydroxy-2-[3-oxo-(4-phenyl-5-(2-chlorophenyl)-3,3a,4,5-tetrahydro-2H-indazol-2-yl)tetrahydro-2H-pyran-3,4-diyl diacetate 14. Its IR spectrum revealed the presence of absorption bands at 3443 cm -1 (OH), 1742 cm -1 (C=O) and an absorption band at 1376 cm -1 due to an out of plane CH 3 . On the other hand, 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide 12 reacted with 5c and 6c in acetone and aqueous potassium hydroxide to afford the corresponding nucleosides derivatives 15 and 16, respectively. The IR spectra of 15 and 16 showed absorption bands at 3315-3314 cm -1 (br) and 1655-1648 cm -1 due to OH/NH and C=O groups, respectively. Spectral results showed that the electron withdrawing character of 4-chlorophenyl and the furan ring at positions 4 and 6 in the pyridine ring of the nucleosides 15 and 16 were helpful in the partial hydrolysis of the cyan group at position 3 of the pyridine moiety as evidenced by the fact that no cyano stretching absorption band was observed in the IR. The 1 H-NMR spectrum of 15 (DMSO-d 6 ) showed signals at δ 4.41 ppm due to NH protons, an aromatic multiplet at δ 6.68-7.90 ppm, two doublets at δ 8.05 and 8.07 ppm due to OH protons, triplets at δ 3.3, 3.41, 3.44 ppm due to CH 2 protons and a singlet at 8.07 ppm due to NH 2 protons. Its mass spectrum showed a molecular ion peak at m/z 497.45. The 1 H-NMR spectrum of 16 (DMSO-d 6 ) showed signals at δ1.97 and 2.49 ppm as two singlets arising from the two COCH 3 groups at C 2 ' , C 3 ' , a singlet signal at δ 6.55 ppm due to proton in the pyridine moiety, two doublets at δ 2.44 and 3.44 ppm due to two anomeric protons at C 2 ' and C 3 ' , a multiplet at δ 6.82-7.94 ppm for the aromatic protons and two signals, one at δ 8.12 ppm for the NH 2 and at 9.75 ppm due to the OH proton at C 4 ' . Its mass spectrum showed the molecular ion peak at m/z 511.5.

Antimicrobial activity
Previously untested compounds were evaluated for antimicrobial activity against eight strains of microorganisms using the agar diffusion technique. The tested compounds were screened against two Grampositive bacteria, Staphylococcus aureus (RCMB 000106) Bacillus subtilis (RCMB 000107); two Gram-negative bacteria, Pseudomonas aeruginosa (RCMB 000102), Escherichia coli (RCMB 000103) and four fungi, Aspergillus fumigatus (RCMB 002003), Geotrichum candidum (RCMB 052006), Candida albicans (RCMB 005002) and Syncephalastrum racemosum (RCMB 005003) by the disk diffusion method. Penicillin G. Streptomycin were used as positive control for bacterial strains while, Itraconazole and Clotrimazole were used as positive controls for the fungi strains. The investigation of antibacterial screening data revealed that compounds 5c and 10e were the most potent towards the Grampositive bacteria S. aureus and B. subtilis. Compounds 6c, 10e and 11b showed good to moderate activity against Gram-positive bacteria S. aureus and B. subtilis. Compound 2 was less potent, while 7b, 10a were inactive. As for the bacterial inhibition of the Gram-negative bacteria, the screening data showed that compounds, 5c and 7a were the most potent against E. coli. Compounds 2, 6c and 11b showed a relatively poor inhibition towards E. coli. All the tested analogs showed no activity against P. aeruginosa. Similarly, compounds 7b and 10a,c,e were inactive against the Gram-negative bacteria P. aeruginosa or E. coli. The bacterial zone of inhibition values are given in Table 1.
Minimum inhibitory concentrations (MICs) were determined by the broth dilution technique. The nutrient broth, which contained logarithmic serially two fold diluted amounts of test compound, and controls were inoculated with approximately 5 × 10 5 c.f.u./ml of actively dividing bacteria cells. The cultures were incubated for 24 h. At 37°C and the growth was monitored visually and spectrophotometrically. The lowest concentration (highest dilution) required to arrest the growth of bacteria was regarded as minimum inhibitory concentration (MIC). To obtain the minimum bactericidal concentration (MBC), 0.1 ml volume was taken from each tube and spread on agar plates. The number of c.f.u. was counted after 18-24 h of incubation at 35°C. MBC was defined as the lowest drug concentration at which 99.9% of the inoculums were killed. The minimum inhibitory concentration and minimum bactericidal concentration are given in Table 2.

Antifungal studies
Antifungal activity testing was also done by the disk diffusion method [43]. For assaying antifungal activity Aspergillus fumigatus  MIC (µg/ml)=Minimum inhibitory concentration, that is, the lowest concentration of the compound to inhibit the growth of bacteria completely; MBC (µg/ml)=minimum bactericidal concentration, that is the lowest concentration of the compound for killing the bacteria completely.

Cytotoxicity studies
Cytotoxicity tests were performed using compounds 4, 5c, 10c and 11c against three cancer cell lines, breast cancer cell line MCF-7, colon carcinoma cells (HCT), human epidermid/arynx carcinoma cell line (HEp2) by using a modified method [35]. The results (Tables 3 and 4) showed that 4 had slight activity toward the HCT cell line (IC 50 =4.7 µg/ ml) and its activity towards MCF-7 cell line was lower (IC 50 =2.7 µg/ mL). Compound 11c exhibited cytotoxic activity against the HCT cell line (IC 50 10.2 µg/ml) and a higher cytotoxic activity against MCF-7 with IC 50 =20.7 µg/ml. The cytotoxic activity of 5c towards HEP-2 was moderately potent with an IC 50 =10.2 µg/ml while its cytotoxic activity against colon carcinoma cells was very low with an IC 50 =2.1µg/ml. The cytotoxic activity of 10c towards the MCF-7 cell line was relatively weak with an IC 50 =4.8 µg/ml, while the cytotoxicity against HCT cell line was nearly inactive given the observed IC 50 of 0.5 µg/ml. The nucleoside analogs 14, 17a, 18a, and 20b (Table 5) showed cytotoxic activity against HTC and MCF-7 and Hepatocellular carcinoma cells HepG2. The IC 50 of compound 14, with values 0.9 and 1.5 µg/ml, indicates high potency against HCT and MCF-7, respectively [44,45]. The nucleoside analog 17a exerted cytotoxic activity against HepG 2 with IC 50 =2 µg/ml. The nucleoside analog 18a exerted activity against HCT with IC 50 of 19.2 µg/ml which decreased when tested against MCF-7 to an IC 50 =1.1 µg/ml. The nucleoside analog 20b selectively exhibited cytotoxic activity against HCT and MCF-7 cell lines with IC 50 of 16.7 and 14.4 µg/ml respectively.

Conclusion
In summary, we have synthesized a novel series of nucleoside analogs in moderate to high yields. The prepared compounds which contain the pyridine-3-carbonitrile moiety provide better antimicrobial activity against S. aureus, B. subtilis than similar molecules without this functionality. Most of the prepared compounds revealed potential anticancer activity against the colon cancer cell line, Hepatocellular cancer cell line, Breast cell line and epidermis/larynx cancer cell line. Compounds 4, 5c, 10c, 11c, 14, 17a, 18a and 20b exhibited good antitumor activity when compared with the reference drug. MIC (µg/ml)=Minimum inhibitory concentration, that is the lowest concentration of the compound to inhibit the growth of fungus completely. MFC (µg/ml)=Minimum fungicidal concentration, that is, the lowest concentration of the compound for killing the fungus completely.