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Synthesis and Antitumor Activity of New 3-Allylseleno-6-alkoxypyridazines against Breast Cancer MCF-7 Cells

  • Received : 2013.12.10
  • Accepted : 2014.01.02
  • Published : 2014.05.20
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Abstract

Keywords

Results and Discussion

As a pharmacologically active group, the allylseleno group was introduced into the pyridazine ring in order to prepare new organoselenium compounds. Dichloropyridazinyl di-selenide 2 was prepared through diselenylation according to the method of Bhasin et al.9,10

Elemental selenium suspended in dimethylformamide reacts with 99% hydrazine hydrate in the presence of NaOH at room temperature to give dark green solution containing diselenide anion. The diselenide anion thus formed reacts in situ with the 3,6-dichlorpyridazine 1. Compound 2 was syn-thesized according to the previously described method.8a,b

Dichloropyridazinyl diselenide 2 can be quantitatively reduced to chloropyridazinyl selenolate anion using hydra-zine hydrate at room temperature in the presence of NaOH in THF. The anion thus formed reacts readily with allyl bromide to give the 3-allylseleno-6-chloropyridazine 3. The 3-allylseleno-6-chloropyridazine 3 is an important inter-mediate for allylselenopyridazine analogs. These reactions were carried out in the presence of the phase-transfer cata-lyst NH4Cl (or TBAB) in an effort to improve the efficiency of the reaction.

Table 1.The optimal conditions alkoxylation for target compounds 4a-4h and their anti-proliferative activity against cell lines MCF-7 compared to 5-FU in CCK-8 assays

The pyridazine NMR peak of 3 appeared at 7.25 and 7.41 ppm, and the allyl peak appeared at 4.0, 5.07, 5.29 and 6.1 ppm. The pyridazine 13C NMR peak appeared at 118.76, 133.90, 155.16 and 158.59 ppm, and the allyl peak appeared at 29.15, 127.71 and 131.24 ppm.

We designed organoselenium analogs of pyridazine in which an allylselenium moiety was introduced at the 3-position of the pyridazine nucleus and the 6-position was substituted with an oxygen (O) atom. Novel 3-allylseleno-6-alkoxypyridazines 4a-4h were prepared through the alkoxyl-ation of 3-allylseleno-6-chloropyridazine 3 and the related alcohol in order to discover potential antitumor candidates. In Table 1, we summarize the optimal conditions for compounds 4a-4h. The yields of compounds 4a-4h were 18-84% and those were identified by NMR.

For investigation of the potential anti-cancer activity of the 8 synthetic compounds, the growth-inhibitory effect of the synthetic compounds was examined against breast cancer (MCF-7) cells.11 CCK-8 assays were performed on cells treated with various concentrations of the compounds.12 5-Fluorouracil (5-FU), which has previously been shown to have anti-proliferative against MCF-7 cells was used as a positive control. We expect that synthesized compounds and 5-FU have similar mechanism of action. The IC50 values for these compounds were determined from the concentration range used in this study.

Of the 8 target compounds, four compounds (4a, 4c, 4d and 4e) inhibited the growth of breast (MCF-7) cells at standard concentrations (6.25, 25, 100, and 400 μg/mL) (Figure 2). 5-FU, a positive control, showed inhibitory effects on growth of MCF-7 cells at standard concentrations. We further investigated the anti-proliferative activity of compound 4e, which caused greater inhibition of cell growth than the other compounds. Compound 4e markedly inhibited MCF-7 cell growth at IC50 (218.55 μM) in a dose-dependent manner at a low concentration (6.25 μg/mL). Eight compounds (4a-4h) showed higher potency than 5-FU at IC50 (3670.59 μM) in inhibiting the growth of MCF-7 cells, suggesting the potential anticancer activity of these compounds (Table 1). The results indicated that compound 4e had the highest activity towards MCF-7 cells.

Figure 2.Antitumor activity of synthesized compounds (4a-4h) in MCF-7 breast cancer cells.

Finally, a new series of 3-allylseleno-6-alkoxylpyridazines 4a-4h was synthesized by synthetic route from 3,6-dichloro-pyridazine for development of new anticancer agents. These new compounds showed anti-proliferative activities against breast cancer (MCF-7) cells in CCK-8 assays, and could be promising candidates for chemotherapy of carcinomas. Among 8 synthesized compounds for inhibiting the growth of these cell lines, compound 4e showed the highest potency. This result suggests the potential anticancer activity of compound 4e.

 

Experimental

Chemicals. Chemicals were supplied by Aldrich, Sigma, Merck, and Tokyo Kasei. Melting points were determined in open capillary tubes on a Büchi 535 melting point apparatus and were uncorrected. NMR spectra were recorded using a Bruker 300 MHz NMR spectrometer. IR spectra were recorded on a Perkin-Elmer 16F PC FT-IR spectrometer using NaCl discs and pellets. Mass fragmentations were recorded using an Agilent 6890 GC and 5973 MS.

Materials and Methods for Bioassays. Cell Line Culture Conditions. MCF-7 breast cancer cells were purchased from the ATCC (Manassas, USA) and maintained at 37 °C in a humidified atmosphere, with 5% CO2, in MEM (Gibco-BRL Inc.) medium supplemented with 10% fetal bovine serum (Gibco-BRL Inc., Korea).

Anti-proliferative CCK-8 (cell counting kit-8) Assays.12 The cytotoxic activity of the compounds was determined in vitro using the CCK-8 assay kit (Dojindo, Korea). Human breast cancer cells were seeded in 96-well plates at densities of 5000 cells/well with five replicates for each drug concent-ration and maintained at 37 °C in a 5% CO2 humidified incubator for 24 h. Control cells were treated with dimethyl sulfoxide (DMSO) equal to the highest percentage of solvent used in the experimental conditions. 5-FU was used as a positive control. The MCF-7 cells were then treated with various concentrations of synthetic compounds (the final concentrations of 4a-4h were 6.25, 25, 100, and 400 μg/mL) for 24 h; 10 μL of Cell Counting Kit-8 solution were added to each well (containing 100 μL), and the plates were further incubated for 2.5 h. The absorbance was measured at 450 nm using a micro ELISA reader (ASYS Biotech, Cambridge, BK). The cell viability ratio was calculated as follows: (test group A450/control group A450) × 100%. IC50 values were determined from three independent experiments.

3-Allylseleno-6-chloropyridazine 3. To a vigorously stirred mixture of powdered sodium hydroxide (1.03 g, 26 mmol), tetrabutylammonium bromide (TBAB) (0.34 g, 1.04 mmol), dichloropyridazinyl diselenide 2 (2 g, 5.2 mmol) and distilled THF (30 mL), 99% hydrazine hydrate (0.054 mL, 1.73 mmol) was added dropwise at rt. Stirring was continued for additional 45 min. Allyl bromide (0.86 mL, 10.4 mmol) was added dropwise at 0 °C. Stirring was continued for addi-tional 1 h at 0 °C. Upon completion, the reaction was stopp-ed and the solvent was evaporated under reduced pressure. The residue was dissolved in ethyl acetate (50 mL). The organic layer was washed with water (50 mL × 4) and then dried over anhydrous Na2SO4. After solvent evaporation, the residue was purified by silica gel column chromatography (3:1 v/v, hexane:EtOAc) to afford 3 as a pale yellow liquid. Yield: 68%, mp 78-80 °C. 1H NMR (CDCl3) δ 7.41 (d, J = 9 Hz, 1H, pyridazine), 7.25 (d, J = 9 Hz, 1H, pyridazine), 6.10-6.00 (m, 1H, CH, allyl), 5.29 (d, J = 17.5 Hz, 1H, =CH2, allyl), 5.07 (d, J = 10.3 Hz, 1H, =CH2, allyl), 4.00 (d, J = 7.5 Hz, 2H, SeCH2). 13C NMR (CDCl3) δ 158.59, 155.16, 133.90, 118.76 (pyridazine), 131.24, 127.71, 29.15 (allyl). FT-IR (NaCl) cm−1 3032 (aromatic), 1557 (N=N), 765 (C-Se), 706 (C-Cl). GC-MS m/z (%) 234 (M+) 219.0 (100.00), 217.0 (48.7), 221.0 (43.9), 153.1 (23.9), 118.1 (23.5).

General Procedure for 3-Allylseleno-6-alkoxypyrida-zines 4a-4h. To a stirred mixture of powdered sodium hydroxide (83 mg, 2.06 mmol), water and the appropriate absolute alcohol (30 mL), 3-allylseleno-6-chloropyridazine 3 (0.48 g, 2.06 mmol) was added as powder at rt. Metal sodium and related alcohols were useful reagents for gene-rating the alkoxide in the case of long chain alcohols such as butanol, pentanol, hexanol, isopropanol and isobutanol as shown in Table 1. The mixture was stirred for 2-93 h at rt~reflux. Upon completion, the reaction was stopped and the excess alcohol was evaporated under reduced pressure. The residue was dissolved with ethyl acetate (50 mL). The organic layer was washed with water (50 mL × 2) and then dried over anhydrous Na2SO4. After solvent evaporation, the residue was purified by silica gel column chromatography (hexane: EtOAc) to afford 4.

3-Allylseleno-6-methoxypyridazine (4a): Yield: 61%, mp 31-32 ℃. 1H NMR (CDCl3) δ 7.32 (d, J = 9 Hz, 1H, CH, pyridazine), 6.80 (d, J = 9 Hz, 1H, CH, pyridazine), 6.12-6.03 (m, 1H, CH, allyl), 5.24 (d, J = 17.7 Hz, 1H, =CH2, allyl), 5.04 (d, J = 10.2 Hz, 1H, =CH2, allyl), 4.10 (s, 3H, OCH3, methyl), 3.96 (d, J = 7.2 Hz, 2H, SeCH2). 13C NMR (CDCl3) δ 163.98, 151.77, 131.83, 117.60 (pyridazine), 134.23, 117.54, 28.75 (allyl), 54.71 (OCH3). FT-IR (NaCl) cm−1 3080 (aromatic), 2947 (aromatic), 1584 (N=N), 1398 (CH3), 1008, 916 (allyl double band), 833 (CSe). GC-MS m/z (%) 230 (M+), 215.0 (100.0), 230.0 (71.3), 213.0 (50.5), 228.0 (36.0), 149.1 (29.7).

3-Allylseleno-6-ethoxypyridazine (4b): Yield: 18%, Oil. 1H NMR (CDCl3) δ 7.32 (d, J = 9.1 Hz, 1H, CH, pyrida-zine), 6.77 (d, J = 9.12 Hz, 1H, CH, pyridazine), 6.12-6.03 (m, 1H, CH, allyl), 5.23 (d, J = 16.2 Hz, 1H, =CH2, allyl), 5.04 (d, J = 9.9 Hz, 1H, =CH2, allyl), 4.53 (q, J = 7.1 Hz, 2H, OCH2, ethyl), 3.95 (d, J = 8.5 Hz, 2H, SeCH2), 1.44 (t, J = 7.1 Hz, 3H, CH3, ethyl). 13C NMR (CDCl3) δ 164.16, 151.79, 132.22, 118.32 (pyridazine), 134.64, 117.94, 28.94 (allyl), 63.56 (OCH3), 14.89 (ethyl). FT-IR (NaCl) cm-1 3081 (aromatic), 2924 (aromatic), 1583 (N=N), 1306 (CH2), 1028 (CH3), 908, 839 (allyl double band), 839 (CSe). GC-MS m/z (%) 229.0 (100.0), 135.0 (50.7), 227.0 (50.5), 201.0 (47.1), 199.0 (23.0).

3-Allylseleno-6-propoxypyridazine (4c): Yield: 84%, mp 53-54 ℃. 1H NMR (CDCl3) δ 7.32 (d, J = 9 Hz, 1H, CH, pyridazine), 6.78 (d, J = 9 Hz, 1H, CH, pyridazine), 6.12-6.03 (m, 1H, CH, allyl), 5.23 (d, J = 18 Hz, 1H, =CH2, allyl), 5.03 (d, J = 9.9 Hz, 1H, =CH2, allyl), 4.42 (t, J = 6.9 Hz, 2H, OCH2, propyl), 3.95 (d, J = 7.5 Hz, 2H, SeCH2), 1.90-1.78 (m, 2H, CH2, propyl), 1.05 (t, J = 7.5 Hz, 3H, CH3, propyl). 13C NMR (CDCl3) δ 163.98, 151.35, 131.83, 117.56 (pyrida-zine), 134.28, 117.68, 28.56 (allyl), 68.94, 22.18, 10.42 (prop-yl). FT-IR (NaCl) cm-1 3086 (aromatic), 2962 (aromatic), 1590 (N=N), 1382 (CH2), 1188 (CH3), 926, 839 (allyl double band), 839 (CSe). GC-MS m/z (%) 258 (M+), 201.0 (100.0), 258.1 (78.9), 243.0 (71.7), 135.1 (57.3), 199.0 (49.2).

3-Allylseleno-6-butoxypyridazine (4d): Yield: 24%. Oil. 1H NMR (CDCl3) δ 7.31 (d, J = 9 Hz, 1H, pyridazine), 6.77 (d, J = 9 Hz, 1H, pyridazine), 6.12-6.03 (m, 1H, CH, allyl), 5.23 (d, J = 16.8 Hz, 1H, =CH2, allyl), 5.04 (d, J = 9.9 Hz, 1H, =CH2, allyl), 4.46 (t, J = 6.6 Hz, 2H, OCH2), 3.95 (d, J = 7.5 Hz, 2H, SeCH2), 1.84-1.75 (m, 2H, CH2, butyl), 1.52-1.44 (m, 2H, CH2, butyl), 0.97 (t, J = 7.4 Hz, 3H, CH3, butyl). 13C NMR (CDCl3) δ 164.36, 151.74, 132.20, 118.06 (pyridazine), 134.67, 117.94, 28.93 (allyl), 67.61, 31.27, 19.56, 14.20 (butyl). FT-IR (NaCl) cm−1 3080 (aromatic), 2958 (aromatic), 1583 (N=N), 1379 (CH2), 1129 (CH3), 988, 915 (allyl double band), 832 (CSe). GC-MS m/z (%) 272 (M+) 257.0 (100.0), 201.0 (96.4), 135.1 (73.1), 255.1 (50.1), 199.0 (47.7).

3-Allylseleno-6-pentoxypyridazine (4e): Yield: 47%. mp 46-48 °C. 1H NMR (CDCl3) δ 7.29 (d, J = 9.1 Hz, 1H, pyridazine), 6.74 (d, J = 9.1 Hz, 1H, pyridazine), 6.12-5.98 (m, 1H, CH, allyl), 5.21 (d, J = 18 Hz, 1H, =CH2, allyl), 5.01 (d, J = 9.9 Hz, 1H, =CH2, allyl), 4.42 (t, J = 6.7 Hz, 2H, OCH2), 3.92 (d, J = 7.5 Hz, 2H, SeCH2), 1.84-1.74 (m, 2H, CH2, pentyl), 1.43-1.32 (m, 4H, CH2, pentyl), 0.90 (t, J = 6.9 Hz, 3H, CH3, pentyl). 13C NMR (CDCl3) δ 164.12, 151.51, 131.97, 117.83 (pyridazine), 134.44, 117.71, 28.71 (allyl), 67.66, 28.71, 28.26, 22.58, 14.19 (pentyl). FT-IR (NaCl) cm−1 3080 (aromatic), 2952 (aromatic), 1589 (N=N), 1380 (CH2), 1128 (CH3), 995, 919 (allyl double band), 840 (CSe). GC-MS m/z (%) 286 (M+), 201.0 (100.0), 217.1 (78.9), 286.1 (59.5), 199.0 (50.3), 153.1 (43.8).

3-Allylseleno-6-hexoxypyridazine (4f): Yield: 24%. Oil, 1H NMR (CDCl3) δ 7.31 (d, J = 9.1 Hz, 1H, pyridazine), 6.76 (d, J = 9.1 Hz, 1H, pyridazine), 6.12-6.00 (m, 1H, CH, allyl), 5.23 (d, J = 16.9 Hz, 1H, =CH2, allyl), 5.03 (d, J = 9.9 Hz, 1H, =CH2, allyl), 4.45 (t, J = 6.7 Hz, 2H, OCH2), 3.95 (d, J = 7.3 Hz, 2H, SeCH2), 1.85-1.78 (m, 2H, CH2, hexyl), 1.45-1.26 (m, 6H, CH2, hexyl), 0.90 (t, J = 4.6 Hz, 3H, CH3, hexyl). 13C NMR (CDCl3) δ 164.15, 151.52, 132.00, 117.84 (pyridazine), 134.45, 117.71, 28.99 (allyl), 67.70, 31.70, 28.74, 25.80, 22.76, 14.20 (hexyl). FT-IR (NaCl) cm−1 3080 (aromatic), 2954 (aromatic), 1579 (N=N), 1380 (CH2), 1129 (CH3), 995, 916 (allyl double band), 833 (CSe). GC-MS m/z (%) 300 (M+), 234.2 (100.0), 150.1 (66.4), 85.2 (54.7), 117.1 (40.0), 55.1 (23.4).

3-Allylseleno-6-isopropoxypyridazine (4g): Yield: 38%, mp 53-54 °C, 1H NMR (CDCl3) δ 7.31 (d, J = 9.1 Hz, 1H, CH, pyridazine), 6.72 (d, J = 9.1 Hz, 1H, CH, pyridazine), 6.12-6.03 (m, 1H, CH, allyl), 5.55-5.46 (m, 1H, OCH2, isopropyl), 5.23 (d, J = 16.9 Hz, 1H, =CH2, allyl), 5.03 (d, J = 9.9 Hz, 1H, =CH2, allyl), 1.40 (d, J = 6.1 Hz, 6H, CH3×2, isopropyl). 13C NMR (CDCl3) δ 163.79, 151.30, 132.26, 118.52 (pyridazine), 134.64, 117.90, 28.91 (allyl), 70.20, 22.27, 22.15 (isopropyl). FT-IR (NaCl) cm−1 3081 (aromatic), 2925 (aromatic), 1582 (N=N), 1028, 908 (allyl double band), 838 (CSe). GC-MS m/z (%) 256 (M+), 201.0 (100.0), 258.0 (70.1), 199.0 (63.2), 135.1 (40.1), 243.0 (37.6).

3-Allylseleno-6-isobutoxypyridazine (4h): Yield: 31%. Oil, 1H NMR (CDCl3) δ 7.33 (d, J = 9.0 Hz, 1H, pyridazine), 6.79 (d, J = 9.0 Hz, 1H, pyridazine), 6.14-6.00 (m, 1H, CH, allyl), 5.23 (d, J = 16.9 Hz, 1H, =CH2, allyl), 5.03 (d, J = 9.9 Hz, 1H, =CH2, allyl), 4.24 (d, J = 6.6 Hz, 2H, OCH2), 3.95 (d, J = 7.5 Hz, 2H, SeCH2), 2.20-2.07 (m, 1H, CH, isobutyl), 1.02 (d, J = 6.7 Hz, 6H, CH3×2, isobutyl). 13C NMR (CDCl3) δ 164.41, 151.70, 132.18, 118.00 (pyridazine), 134.65, 117.88, 28.88 (allyl), 73.87, 28.18, 19.52 (isobutyl). FT-IR (NaCl) cm−1 3081 (aromatic), 2925 (aromatic), 1583 (N=N), 1006, 911 (allyl double band), 833 (CSe). GC-MS m/z (%) 272 (M+), 201.0 (100.0), 272.1 (65.8), 199.0 (60.8), 257.0 (50.8), 135.1 (42.0).

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