Synthesis and anti-proliferative activity of pyridine O-galactosides and 4-fluorobenzoyl analogues

Introduction

Nucleoside analogues have been explored due to their structural similarity to the naturally occurring nucleosides, which are the fundamental building blocks of many biological systems. The recent interest in nucleosides as biologically active agents stimulates the development of novel compounds targeting common cancers [1]. As a result, synthetic nucleosides are used in chemotherapy, and there is an intertwined relationship between natural and synthetic nucleosides [2]. Cytotoxic nucleosides interfere with nucleic acid synthesis, and many of these are quite promising agents for cancer therapies with different mechanisms of action. In particular, 4-amino-3-fluoro-1-(β-d-ribofuranosyl)-2(1H)-pyridone inhibits the growth of HL-60 lymphoid leukemia cells with IC₅₀ = 1.07×10^-5m (Figure 1), whereas its 2′-deoxy analogue is active against lymphoid leukemia L1210 cells [3–5].

Figure 1

Fluoropyridine analogues used against lymphoid leukemia.

In our previous study [6–8], we reported the synthesis and apoptotic activity of pyrimidine, pyrazoline, and pyridine nucleosides. The goal of this work was to synthesize new pyridine analogues and to confirm their structures by analyzing their NMR properties. The anti-proliferative activities in HL-60 leukemia cells of the resultant compounds were studied.

Results and discussion

A series of 2(1H)-pyridone derivatives 1(Schemes 1–3) was synthesized previously in yields of 84–96% [8, 9]. In this work, alkylation of a particular compound 1′ with iodoethane under basic conditions yielded a mixture of N-ethylpyridone 2a and 2-ethoxypyridine 2b (Scheme 1). The isomeric mixture of 2a and 2b was separated using column chromatography [10–12]. The ¹H NMR data obtained for isomers 2a and 2b indicate different chemical shifts for the pyridine H-5 atom [13–15]. For the N-ethyl isomer, the chemical shift of the pyridine H-5 atom is observed at δ 6.53, whereas the H-5 signal for the ethoxy isomer 2b is shifted downfield to δ 7.76 (see the Supplementary data for 2a and 2b). This difference in chemical shifts was used to assign the structures to O-nucleosides 3, 4, and their O-benzoyl analogues 5 synthesized as part of this work (Schemes 2 and 3).

Scheme 1

Synthesis of N-ethylpyridone 2a and 2-ethoxypyridine 2b.

Scheme 2

Synthesis of pyridine O-galactosides 3 and 4.

Scheme 3

Synthesis of O-(4′-fluorobenzoyloxy)pyridine analogues 5a–d.

Treatment of potassium salts of 2-pyridones 1 [6–8] with 1-bromo-2,3,4,6-tetra-O-acetyl-α-d-galactopyranoside yielded the corresponding acetyl-protected O-β-nucleosides 3a–e. Deacetylation of products 3a–d under standard conditions provided the respective O-β-nucleosides 4a–d(Scheme 2). The β-configuration of 3 and 4 is consistent with the large spin-spin coupling constant (J_{H-1″-H-2″}>7.00 Hz) for protons H-1″ and H-2″ in these compounds [8]. The chemical shift of pyridine H-5 proton of the obtained nucleosides 3 and 4 is observed in the range of δ 7.65–8.30, which indicates the O-substitution (see the Supplementary data Figs. 5–22).

The 2-(4′-fluorobenzoyloxy) analogues 5a–d were synthesized as shown in Scheme 3. The reaction of substrates 1 with 4-fluorobenzoyl chloride yielded a single isomer 5a–d in each case. The O-substitution in 5a–d was determined as discussed above. In addition, the infrared spectra for 5show absorption in the range of 1750–1770 cm^-1 for the benzoyl carbonyl group. In the ¹³C NMR spectrum, the characteristic chemical shift for this group appears in the region of δ 157.4–158.3 (see the Supplementary data Figs. 23–30).

The effects of pyridine galactosides 3 and 4 and their benzoyl analogues 5as anti-proliferative agents of the human promyelotic leukemia (HL-60) cell lines were evaluated. The results of the MTT cell proliferation assay show that compound 4c has the highest anti-proliferation activity among all synthesized nucleosides 3 and 4. Interestingly, non-nucleosides 5a–d were found to have even higher anti-proliferation activities than the corresponding nucleosides 3 and 4. Interestingly, the non-nucleoside analogue 5a shows the highest activity among all synthesized compounds [16–18].

Conclusions

A new series of pyridine O-galactoside 3, 4, and 4-fluorobenzoyl analogues 5 were synthesized. All compounds show some anti-proliferative activities, with 5a having the highest activity.

Experimental

All air-sensitive materials were handled under a nitrogen atmosphere. Melting points were determined in Pyrex capillaries on a Gallenkamp apparatus. Infrared spectra were recorded with a Thermo Nicolet Nexus 470 FT-IR spectrometer in potassium bromide disks. ¹H NMR and ¹³C NMR spectra were obtained on Varian 200 or 400 instruments. Optical rotations were measured with a Perkin-Elmer digital polarimeter at 589 nm (sodium D line) in a 1-dm cell. Thin-layer chromatography (TLC) was carried out on precoated Merck silica gel F₂₅₄ plates and ultraviolet light was used for visualization. Column chromatography was performed using a Merck silica gel. Micro analyses (C, H, and N) were performed on a Flash-EA-1112 series analyzer. The reagents were purchased from Aldrich and used without further purification.

Reagents, tissue culture, and culture conditions

All compounds tested were dissolved in DMSO (200-mm solutions) and subsequently diluted in the culture media before treatment of the culture cells. Human promyelotic leukemia HL-60 cells were obtained from the American Type Culture Collection and Roche Molecular Biochemical, USA. Cells were grown in DMEM medium (GIBCO-BRL) supplemented with 20% fetal calf serum (GIBCO-BRL). The cells were maintained at 37°C in a 5% CO₂ incubator. After reaching confluence, the cells were sub-cultured into 96-well plates, allowed to grow for 24 h, and treated with various concentrations of compounds 3, 4, and 5.

MTT cell proliferation assay [16–18]

Cells were plated in 96-well plates at a density 4×10³ cells/well/100 μL of the appropriate culture medium and treated with compounds 3–5 at concentrations of 100 and 200 μm for 24 h. In parallel, cells were treated with 0.1% of DMSO as a control. A MTT [3-(4′,5′-dimethylthiazol-2′-yl)-2,5-diphenyltetrazolium bromide] assay was performed according to the manufacturer’s instructions provided by Roche. After adding MTT, the yellow mixture was incubated for 1–4 h, followed by spectrophotometric measurements at 450 nm in the 96-well plates. Absorbance is directly proportional to the number of living cells in culture. The results are given in Figure 2.

Figure 2

Cell viability of HL-60, treated with different doses (100 and 200 μm) of compounds 3, 4, and 5 for 24 h at 37°C, using MTT cell proliferation assay.