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added to the solution of 5-iodo-acetyl protected nucleosides (3) 1H, J ¼ 4.0 Hz, 50-OH), 4.10–4.14 (m, 1H, 30-H), 4.00–4.04
(1.14 mmol) which dissolved in anhydrous and peroxide-free (m, 1H, 20-H), 3.89–3.90 (m, 1H, 40-H), 3.56–3.72 (m, 2H, 50-H);
1,4-dioxane (30 mL) under argon atmosphere, the mixture was 13C NMR (DMSO-d6, 500 MHz) d: 187.52 (C-4), 172.04 (C-2),
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heated at 90 C and reuxing for 3 h (TLC tracking reaction). 147.36 (C-200), 136.91 (C-500), 135.73 (C-6), 127.09 (C-400),
Aer cooling down, the black solid was ltered through celite to 126.23 (C-300), 117.61 (C-5), 89.80(C-40), 85.20 (C-10), 74.87 (C-
remove the solvent. Silica gel chromatography (10% CH3OH/ 30), 69.57 (C-50), 60.32 (C-20), HRMS: [M + Na]+ 365.0244,
CH2C12) yielded white solid compound with yield of around calculated for C13H14N2O5S2Na; found 365.0249. UV-vis (in
93%, namely 5-(200-thienyl)-acetyl protected nucleoside (4). Mp: CH3CN): lmax/nm: 239, 287, 356. IR (lm)/cmꢀ1: 3344.3 (–OH),
178–181 ꢁC. 1H NMR (400 MHz, DMSO-d6) d: 11.83 (brs, 1H, 1699.7 (C]O), 1519.0, 1280.8 (C]C), 1600.2 (C5]C6), 1122.6
NH), 8.17 (s, 1H, 6-H), 7.46–7.50 (m, 2H, 500-H, 300-H), 7.07 (m, (C]S).
1H, 400-H), 5.94–5.95 (d, 1H, J ¼ 4.0 Hz, 10-H), 5.57–5.60 (t, 1H, J
¼ 12.0 Hz, 30-H), 5.34–5.39 (m, 1H, 20-H), 4.25–4.36 (m, 3H, 40-H,
2.4. Toxicity test on colon cancer cells
50-H), 2.04–2.06 (m, 9H, CH3); 13C NMR (DMSO-d6, 500 MHz) d:
The MTT colorimetric method and ow cytometry were
170.49, 169.83, 169.79 (3ꢂ–C]O), 137.27 (C-4), 126.92 (C-2),
126.71 (C-200), 124.02 (C-500), 109.51 (C-6), 89.28 (C-400), 79.75
(C-300), 79.64 (C-5), 72.51 (C-40), 72.44 (C-10), 70.07 (C-30), 69.95
(C-50), 63.44 (C-20), 20.97, 20.77, 20.71 (3ꢂ–CH3); HRMS: [M +
Na]+ 475.0790, calculated for C19H20N2O9SNa; found 475.0793.
UV-vis (in CH3CN): lmax/nm: 262.0, 304.0. IR (lm)/cmꢀ1: 1748.0
(C]OCH3), 1717.5 (C]O), 1459.4, 1374.3 (NH), 1571.3, 1414.0,
1299.8 (C]C), 1627.47 (C5]C6).
employed to analyze the anti-tumor activity of 4-thio-5-(200-
thienyl)uridine on mice colon cancer cell line MC-38 and
human colon cancer cell line HT-29, which were purchased
from the American Type Culture Collection.
2.4.1. Cell antiproliferative activity assay. The proliferation
inhibitory activities of the synthesized target compounds was
evaluated using mouse and human colon tumor cell lines (MC-
38 and HT-29) by the MTT method in vitro. MC-38 and HT-29
cells were cultured in DMEM and RPMI1640 medium, 10%
fetal calf serum, 100 mg mLꢀ1 penicillin, and 100 mg mLꢀ1
streptomycin at 37 ꢁC under 5% CO2, respectively. Then the
cells were seed into 96-well plate at 1 ꢂ 104 cells per well and co-
incubated with 4-thio-5-(200-thienyl)uridine under different
concentrations (nal concentration: 100 mM, 200 mM, 300 mM,
400 mM and 500 mM) for 24 h, respectively. At the end of this
time interval, 20 mL (5 mg mLꢀ1) MTT (3-(4,5 dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide) was added to each well,
and aer incubation at 37 ꢁC for 4 h the MTT solution was
removed and 100 mL of dimethylsulfoxide (DMSO) was added to
dissolve the crystals. The absorbance of each well was measured
at 570 nm.
2.4.2. Cell cycle distribution and apoptosis analysis by ow
cytometry detection. Cell cycle distribution and apoptosis
analysis was conducted according to the reported procedure.24
Briey, the HT-29 cells were inoculated into a 6-well cell culture
plate at 5 ꢂ 105 cells per well and cultured overnight. Then, 2
mL of the culture medium containing different concentrations
of 4-thio-5-(200-thienyl)uridine were added to each well respec-
tively and co-incubated for another 48 hours. Aerwards, the
cells oating in the supernatant were combined with the
adherent fraction, washed with phosphate buffered saline (PBS)
thrice, xed with cool ethanol, treated with RNasꢁe, and then
stained with propidium iodide (50 mg mLꢀ1) at 37 C in a dark
environment. The samples were immediately analyzed by ow
cytometry.
2.3.4. 4-Thio-5-(200-thienyl)-acetyl protected nucleoside (5).
5-(200-Thienyl)-acetyl protected nucleoside (4) (2.32 mmol) were
dissolved in peroxide-free 1,4-dioxane (50 mL), then P2S5 (1.0
g, 4.50 mmol) was added. The mixture was reuxed for 3–4 h
when TLC analysis showed complete disappearance of the
starting material. The solvent was removed under reduced
pressure and the residue was treated with CH2Cl2 for several
times. The residue was separated on a silica gel column,
eluting with petroleum : ethyl acetate (3 : 2, v/v) to give the title
1
ꢁ
compound (5) at yield of 56%. Mp: 171–173 C. H NMR (400
MHz, DMSO-d6) d: 13.07 (brs, 1H, NH), 8.05 (s, 1H, 6-H), 7.57–
7.58 (d, 1H, J ¼ 4.0 Hz, 500-H), 7.29–7.30 (d, 1H, J ¼ 4.0 Hz, 300-
H), 7.06–7.08 (t, 1H, J ¼ 4.0 Hz, 30-H), 5.35–5.41 (m, 1H, 20-H),
4.25–4.36 (m, 3H, 40-H, 50-H), 2.07 (m, 9H, CH3); 13C NMR
(DMSO-d6, 500 MHz) d: 188.58 (C-4), 170.43, 169.78, 169.75
(3ꢂ–C]O), 147.07 (C-2), 136.74 (C-200), 136.41 (C-500), 127.11
(C-6), 126.34 (C-400), 117.96 (C-300), 89.96 (C-5), 79.97 (C-40),
72.72 (C-10), 69.92 (C-30), 66.83 (C-50), 63.22 (C-20), 20.81, 20.77,
20.74 (3ꢂ–CH3); HRMS: [M + H]+ 467.0741, calculated for
C
19H20N2O8S2; found 469.0727. UV-vis (in CH3CN): lmax/nm:
239, 285, 348. IR (lm)/cmꢀ1: 1747.5 (C]OCH3), 1715.6 (C]
O), 1451.7, 1371.3 (NH), 1522.7, 1227.5, 1427.31 (C]C), 1610
(C5]C6), 1108 (C]S).
2.3.5. 4-Thio-5-(200-thienyl)-nucleoside (6). 4-Thio-5-(200-
thienyl)-acetyl protected nucleoside (5) (2.14 mmol) were sus-
pended in absolute MeOH (60 mL, 1.5 mmol) and saturated
with dry ammonia gas. The mixture was stirred at room
temperature for 4.5 h. The resulting solution was evaporated
under reduced pressure at 35 ꢁC and the residue was puried
on a silica gel column packed in (CH2Cl2 : MeOH 9 : 1, v/v) and
2.5. Statistical analysis
provide a solid product at yields of 45%, namely 4-thio-5-(200- Data were presented as mean ꢃ SD of three independent
thienyl) nucleoside (6). Mp: 185–187 ꢁC. 1H NMR (400 MHz, experiments. One-way analysis of variance (ANOVA) was per-
DMSO-d6) d: 12.89 (brs, 1H, NH), 8.51 (s, 1H, 6-H), 7.47–7.48 formed on the data to assess the impact of the variables on the
(d, 1H, J ¼ 4.0 Hz, 500-H), 7.27–7.28 (m, 1H, 300-H), 6.98–7.00 (m, results. SPSS 13.0 statistical soware (SPSS Inc., Chicago, USA)
1H, 400-H), 5.75–5.76 (d, 1H, J ¼ 4.0 Hz, 10-H), 5.50–5.51 (d, 1H, J was used for the statistical analysis of all data. A p value of
¼ 4.0 Hz, OH), 5.30–5.32 (t, 1H, J ¼ 4.0 Hz, OH), 5.06–5.07 (d, #0.05 was considered to be statistically signicant.
This journal is © The Royal Society of Chemistry 2016
RSC Adv., 2016, 6, 70099–70105 | 70101