ꢀ
A. Huczynski et al. / European Journal of Medicinal Chemistry 90 (2015) 296e301
300
without further purification. CDCl3 spectral-grade solvent weas
stored over 3 Å molecular sieves for several days. TLC was carried
out on precoated plates (TLC silica gel 60 F254, Aluminium Plates
Merck) and spots were detected by illumination with an UV lamp.
All the solvent used in flash chromatography were of HPLC grade
(CHROMASOLV from SigmaeAldrich) and were used as received.
The elemental analysis of 2ae2j was carried out on Vario ELIII
(Elementar, Germany).
5.4. Antiproliferative activity of colchicine and its derivatives
Four human cancer cell lines and one murine normal cell line
were used to evaluate antiproliferative activity of colchicine and its
derivatives: human acute promyelocytic leukaemia (HL-60) and its
vincristine-resistant subline e (HL-60/vinc), human colon adeno-
carcinoma cell lines sensitive and resistant to doxorubicin (LoVo)
and (LoVo/DX) respectively, and also normal murine embryonic
fibroblast cell line (BALB/3T3). The BALB/3T3 cell line was pur-
chased from the American Type Culture Collection (ATCC Rockville,
Maryland, USA), HL-60 cell line e from European Type Culture
Collection by courtesy of Professor Spik and Dr Mazurier (Labora-
tory of Biological Chemistry USTL, Lille, France) and HL-60/vinc,
LoVo and LoVo/DX by courtesy of Prof. E. Borowski (Technical
5.2. Spectroscopic measurements
The 1H, 13C spectra were recorded on a Bruker Avance DRX 600
spectrometer. 1H NMR measurements of 1e13 (0.07 mol dmꢁ3) in
CDCl3 were carried out at the operating frequency 600.055 MHz;
flip angle, pw ¼ 45ꢂ; spectral width, sw ¼ 4500 Hz; acquisition
time, at ¼ 2.0 s; relaxation delay, d1 ¼ 1.0 s; T ¼ 293.0 K and using
TMS as the internal standard. No window function or zero filling
was used. Digital resolution was 0.2 Hz per point. The error of the
chemical shift value was 0.01 ppm. The 13C NMR spectra were
recorded at the operating frequency 150.899 MHz; pw ¼ 60ꢂ;
sw ¼ 19,000 Hz; at ¼ 1.8 s; d1 ¼ 1.0 s; T ¼ 293.0 K and TMS as the
internal standard. Line broadening parameters were 0.5 or 1 Hz.
The error of chemical shift value was 0.1 ppm. All spectra were
locked to deuterium resonance of CDCl3.
ꢀ
University of Gdansk, Poland). All the cell lines are maintained in
the Institute of Immunology and Experimental Therapy (IIET),
Wroclaw, Poland.
Human leukaemia cells were cultured in Iscove medium (IIET,
Wroclaw) containing 10% foetal bovine serum, 2 mM
L-glutamine
(SigmaeAldrich, Germany) and 1 g/100 ml doxorubicin for HL-60/
m
vinc (SigmaeAldrich, Germany). Human colon adenocarcinoma cell
lines were cultured in mixture of OptiMEM and RPMI 1640 (1:1)
medium (IIET, Wroclaw), supplemented with 5% foetal bovine
serum (Thermo Fisher Scientific), 2 mM
pyruvate (SigmaeAldrich, Germany) and 10
L
-glutamine, 1 mM sodium
g/100 ml doxorubicin
The 1H and 13C NMR signals were assigned using 2-D (COSY,
HETCOR, HMBC) spectra shown in the Supplementary Materials. 2-
D spectra were recorded using standard pulse sequences from
Varian and Bruker pulse-sequence libraries. The FT-IR spectra of
2ae2j in the mid infrared region were recorded in KBr.
m
for LoVo/DX (SigmaeAldrich, Germany). Murine embryonic fibro-
blast cells were cultured in Dulbecco medium (Gibco), supple-
mented with 10% foetal bovine serum (Thermo Fisher Scientific)
and 2 mM glutamine (SigmaeAldrich, Germany). All culture media
The ESI (Electrospray Ionisation) mass spectra were recorded on
a Waters/Micromass (Manchester, UK) ZQ mass spectrometer
equipped with a Harvard Apparatus syringe pump. The samples
were prepared in dry acetonitrile (5 ꢃ 10ꢁ5 mol dmꢁ3) with the
addition of acetic acid. The sample was infused into the ESI source
contained antibiotics: 100 U/ml penicillin and 100 mg/ml strepto-
mycin (Polfa-Tarchomin, Poland). All cell lines were cultured during
entire experiment in humid atmosphere at 37 ꢂC and 5% CO2.
5.4.1. The antiproliferative assays in vitro
using a Harvard pump at a flow rate of 20
m
l minꢁ1. The ESI source
Twenty four hours before adding the tested compounds, all cell
lines were seeded in 96-well plates (Sarstedt, Germany) in appro-
priate media with 104 cells per well. All cell lines were exposed to
each tested agent at four different concentrations of the range 100
potentials were: capillary 3 kV, lens 0.5 kV, extractor 4 V. The
standard ESI mass spectra were recorded at the cone voltages: 10
and 30 V. The source temperature was 120 ꢂC and the desolvation
temperature was 300 ꢂC. Nitrogen was used as the nebulizing and
to 0.1 mg/ml for 72 h. Cells were also exposed to the reference drug
desolvation gas at flow-rates of 100 dm3
h
ꢁ1. Mass spectra were
cisplatin (Accord) and doxorubicin (SigmaeAldrich, Germany).
Additionally, all cell lines were exposed to ethanol (solvent used for
tested compounds) (SigmaeAldrich, Germany) at concentrations
corresponding to those present in the tested agents' dilutions. For
adherent cells, sulphorhodamine B assay was performed and MTT
assay for leukaemia cells.
acquired in the positive ion detection mode with unit mass reso-
lution at a step of 1 m/z unit. The mass range for ESI experiments
was from m/z ¼ 300 to m/z ¼ 750.
5.3. Synthesis
5.3.1. General procedure for the synthesis of colchicine derivatives
5.4.2. SRB
(2ae2j)
After 72 h of incubation with tested compounds, the cells were
A
solution of colchicine (400 mg;
1
mmol) and 2-(2-
fixed in situ by gently adding 50 ml per well of cold 50% trichloro-
aminoethoxy)ethanol (1.58 g; 15 mmol) was stirred at reflux for
24 h. Following evaporation, the mixture was dissolved in CH2Cl2
(5 ml) and purified chromatographically on silica gel (Fluka type
60) to give compound 2f with yield 72% as a yellow powder. The
exemplary 2D NMR spectra of compound 2f are included in the
acetic acid TCA (Avantor, Poland) and were incubated at 4 ꢂC for one
hour. Then the wells were washed four times with water and air
dried. Next, 50 ml of 0.2% solution of sulphorhodamine B (Sigma-
eAldrich, Germany) in 1% acetic acid (Avantor, Poland) were added
to each well and the plates were incubated at room temperature for
0.5 h. After incubation time, the unbound dye was removed by
washing plates four times with 1% acetic acid, whereas the stain
bound to cells was solubilized with 10 mM Tris base (Sigma-
eAldrich, Germany). Absorbance of each solution was read at
Synergy H4 photometer (BioTek Instruments, USA) at 540 nm
wavelength.
Supplementary material. 13C NMR (150 MHz, CDCl3)
d ppm:174.9
(CaO), 169.9 (CaO), 154.1 (C), 152.6 (C), 151.4 (C), 150.8 (C), 141.2 (C),
139.0 (CH) 134.4 (C), 130.6 (C), 126.5 (C), 122.9 (C), 108.5 (CH) 107.0
(CH), 72.3 (OCH2), 68.4 (OCH2), 61.4 (OCH3), 61.2 (OCH3), 61.1
(HOCH2), 55.9 (OCH3), 52.4 (CH), 42.3 (NHeCH2), 36.8 (CH2), 29.8
(CH2), 22.5 (CH3). 1H NMR (403 MHz, CDCl3)
d ppm: 8.64 (dd,
J ¼ 20.90, 6.53 Hz,1H), 7.58 (s,1H), 7.58 (m,1H), 7.45 (d, J ¼ 11.21 Hz,
1H), 6.66 (d, J ¼ 11.38 Hz, 1H), 6.55 (s, 1H), 4.73e4.65 (m, 1H), 3.94
(s, 3H), 3.90 (s, 3H), 3.82 (t, J ¼ 5.34 Hz, 2H), 3.76 (m, 2H), 3.64 (mz,
2H), 3.63 (s, 3H), 3.58 (dd, J ¼ 10.69, 5.30 Hz, 2H), 3.29e3.02 (bs,
1H), 2.47 (dd, J ¼ 12.55, 5.67 Hz, 1H), 2.25 (m, 2H), 1.97 (s, 3H).
5.4.3. MTT
Proliferation inhibition of leukaemia cells by tested compounds
was measured by means of MTT assay. Thus, 20
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide solution
ml of 3-(4,5-