Derivatives of thiocolchicine and its applications to CEM cells treatment using 19F Magnetic Resonance ex vivo

Article abstract of DOI:10.1016/j.bioorg.2009.10.002

It was shown, that cultured ex vivo human T-Lymphoblastoid (CEM) cells respond to synthesized thiocolchicine and fluorine thiocolchicine derivatives. The preparation of derivatives with substitution at C-3 and C-7 is described. All compounds were used at concentration from 1 nM to 1000 nM. Inhibitory effects of these compounds were examined in the three-dimensional (3-D) culture and cells morphology during treatment was monitored using 9.4 T MRI system. We performed studies of these compounds in CEM cells ex vivo using ¹H and ¹⁹F Magnetic Resonance Imaging (MRI), ¹⁹F Magnetic Resonance Spectroscopy (MRS), High Performance Liquid Chromatography coupled with Ultra Violet (HPLC-UV) and Electron Impact Mass Spectrometry (EIMS). The results of the multi-technique approach are consistent with the fact that the new derivatives are more efficient than colchicine and thiocolchicine ex vivo. Crown Copyright

Full text of DOI:10.1016/j.bioorg.2009.10.002

Bioorganic Chemistry 38 (2010) 1–6
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Preliminary Communication
Derivatives of thiocolchicine and its applications to CEM cells treatment
1
9
using F Magnetic Resonance ex vivo
Dorota Bartusik a,b,*, Boguslaw Tomanek a,b,c,d, Erika Lattová , Hélène Perreault , Jack Tuszynski f,g,
Gino Fallone
a
e
e
b,d
National Research Council Canada, Institute for Biodiagnostics (West), 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1
Cross Cancer Institute, Medical Physics Department, 11560 University Ave, Edmonton, Alberta, Canada T6G 1Z2
Polish Academy of Sciences, Institute of Nuclear Physics, Radzikowskiego 152, 31-342 Kraków, Poland
University of Alberta, Oncology Department, 11560 University Ave, Edmonton, Alberta, Canada T6G 1Z2
University of Manitoba, Department of Chemistry, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2
Cross Cancer Institute, Experimental Oncology Department, 11560 University Ave, Edmonton, Alberta, Canada T6G 1Z2
University of Alberta, Department of Physics, 11322 – 89 Avenue, Edmonton, Alberta, Canada T6G 2GZ
b
c
d
e
f
g
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 May 2009
Available online 1 November 2009
It was shown, that cultured ex vivo human T-Lymphoblastoid (CEM) cells respond to synthesized thi-
ocolchicine and fluorine thiocolchicine derivatives. The preparation of derivatives with substitution at
C-3 and C-7 is described. All compounds were used at concentration from 1 nM to 1000 nM. Inhibitory
effects of these compounds were examined in the three-dimensional (3-D) culture and cells morphology
during treatment was monitored using 9.4 T MRI system. We performed studies of these compounds in
CEM cells ex vivo using H and F Magnetic Resonance Imaging (MRI), F Magnetic Resonance Spectros-
copy (MRS), High Performance Liquid Chromatography coupled with Ultra Violet (HPLC-UV) and Electron
Impact Mass Spectrometry (EIMS). The results of the multi-technique approach are consistent with the
fact that the new derivatives are more efficient than colchicine and thiocolchicine ex vivo.
Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved.
Keywords:
Thiocolchicine
1
19
19
Human T-Lymphoblastoid
Three-dimensional cell culture
Magnetic Resonance Imaging
High Performance Liquid Chromatography
coupled with Ultra Violet
1
. Introduction
An important tool in cancer chemotherapy is quantitative mon-
known to disrupt the dynamic of cancer cells growth. Advances in
synthetic methods, coupled with continued effort aimed at identi-
fication of cell surface receptors contribute to the growing library
of small-molecule inhibitors of cancer cell growth. Well-known
examples include paclitaxel [1], an antimitotic agent known to sta-
bilize microtubules by binding to b-tubulin [2] and colchicine [3],
an antimitotic agent that binds to a different site of b-tubulin and
inhibits its assembly into microtubules. Colchicine, a major alkaloid
of Colchicum autumnale and Gloriosa superba, is a drug interfering
with microtubule structure both in vitro and in vivo, thereby caus-
ing cells to accumulate in mitotic arrest during the cell cycle [4].
Although the antitumor properties of many colchicinoids have been
well-known [5], only one compound, N-deacetyl-N-methylcolchi-
cine (Colcemid), has been used for the treatment of Hodgkin’s lym-
phoma and chronic granulocitic leukemia [6]. Analogues of
colchicine are currently used in therapy of Mediterranean fever
itoring of the response of tumor to treatment. Currently, tumor
response is monitored by detection of changes in tumor morphol-
ogy, mostly its size. There is a potential in using Magnetic Reso-
nance Imaging (MRI) for monitoring of the therapy as MRI
detects an early response to treatment at morphological level.
Therefore, we used MRI to investigate the visualization of human
tumor cultured in three-dimensional (3-D) form treated with thi-
ocolchicine derivatives by monitoring cell changes.
Inhibitory effects of cancer cells growth are principal targets in
the development of novel therapeutics. Among cellular structures,
required to maintain the growth of normal and malignant cells,
the microtubules play a pivotal role as the template for transcrip-
tion and transactivation of cells. Drugs binding to tubulin are
[
7], Behcet’s disease [8], progressive scleroderma [9] and gout [10].
The main aim of the present study was to determine the impact
Abbreviations: CEM, human T-Lymphoblastoid; EIMS, Electron Impact Mass
Spectrometry; HFB, hollow fiber bioreactor; HPLC-UV, High Performance Liquid
Chromatography coupled with Ultra Violet; MR, Magnetic Resonance; MRI,
Magnetic Resonance Imaging.
of structural properties of selected thiocolchicine derivatives on
CEM cells’ growth. We applied Hollow Fiber Bioreactor (HFB) to
grow 3-D culture and produce high density cell culture suitable
for MRI study. For the visualization of the interactions between
*
Corresponding author. Address: National Research Council Canada, Institute for
Biodiagnostics (West), 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1.
Fax: +1 780 432 8615.
1
19
thiocolchicine derivatives and cells we used H and F MRI as well
1
9
E-mail address: dorota.bartusik@gmail.com (D. Bartusik).
as F Magnetic Resonance Spectroscopy (MRS). Our studies were
0
045-2068/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2009.10.002

2
D. Bartusik et al. / Bioorganic Chemistry 38 (2010) 1–6
aimed at the measurements of the effects of the drug action in cell
N-[(7S)-3-hydroxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-
culture ex vivo.
5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]-N-[(trifluoroacetyl)gly-
cyl]acetamide (5a);
N-[(7S)-1,2-dimethoxy-10-methylsulfanyl-9-oxo-3-(prop-2-
enoxy)-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]-N-[(trifluoroace-
2
. Materials and methods
tyl)glycyl]acetamide (5b);
2
.1. Compounds and reagents
N-[(7S)-3-ethoxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-
,6,7,9-tetrahydrobenzo[ ]heptalen-7-yl]-N-[(trifluoroacetyl)gly-
cyl]acetamide (5c).
5
a
Colchicine (1), N-[(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tet-
rahydrobenzo[a]heptalen-7-yl]acetamide, and other compounds
used to synthesis and cell culture were purchased from Sigma–Al-
drich (Oakville, ON). Major histocompatibility complex (MHC) class
I antibody was purchased from Abcam Inc. (Cambridge, MA).
One millimole of appropriate derivative (3a–c) was dissolved in
methanol (20 mL) with 2 M HCl (10 mL), heated at 90 °C and stir-
red for 24 h. The reaction mixture was cooled, neutralized with
NaHCO
over Na
CH Cl /CH
3
3
and extracted with CH
SO and evaporated. The crystallization was from (1:1)
OH. The yield of deacetylated compound (4a–c) was
2 2
Cl . The obtained extract was dried
2
2
.2. Synthesis of the compounds
2
4
2
2
5
8%, 63% and 71%, respectively.
.2.1. Preparation of thiocolchicine, N-[(7S)-1,2,3-trimethoxy-10-
One millimole of deacetylated compound of (4a–c) and N-triflu-
methylsulfanyl-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-yl]acet-
oroacetylglycine (1 mmol) were dissolved at room temperature
and dichloromethane (6 mL) was added with stirring. N-trifluoro-
acetylglycine prepared accordingly to previously published refer-
ence [11]. Dicyclohexylcarbodiimide (1 mmol) was added to the
suspension. After 2 h the mixture was cooled to 0 °C and filtrated.
Each compound (5a–c) was crystallized from dichloromethane:
ethyl ether (1:1). The yield of (5a–c) was 64%, 67% and 75%,
respectively.
amide (2)
Colchicine (1) (1 mmol) was dissolved in 10 mL of methanol/
dimethylformamide (1:1) at 70–80 °C. The solution was cooled to
room temperature and sodium methanethiolate (2 mmol) was
added. The mixture solution was stirred overnight. Water
(
20 mL) was added, and the reaction mixture was extracted with
CH Cl (10 mL), dried over Na SO and concentrated. Crystalliza-
tion of the residue from ethyl ether/acetone (1:1) gave product
2) with 71% yield.
2
2
2
4
(
2.3. Cell cultures
2
.2.2. Preparation of N-[(7S)-3-hydroxy-1,2-dimethoxy-10-
CEM cells, American Type Culture Collection (Rockville, MD),
methylsulfanyl-9-oxo-5,6,7,9-tetrahydrobenzo[a]heptalen-7-
yl]acetamide (3a)
were maintained in tissue culture flasks and cultured as monolayer
in 20 mL of RPMI media containing 10% Fetal Bovine Serum (FBS).
Ten millimoles of methanol was used to dissolve 1 mmol of thi-
ocolchicine (2) and 30 mL of 0.2 M of hydrochloric acid was added.
The methanol was evaporated, cooled and sodium hydroxide
solution was added until pH value was 11. The resulting alkaline
solution was extracted with chloroform in order to free it from
non-phenolic substances. The sodium hydroxide solution, (color
red), was acidified with hydrochloric acid and was extracted with
chloroform. After drying and evaporation, the yield of (3a) was
5
4
The cells were divided from 5 ꢀ 10 cells/mL to 2.5 ꢀ 10 cells/mL
two times per week. When the number of cells in the culture flask
6
reached 5–6 ꢀ 10 cells/mL the culture was harvested and then
inoculated into six Hollow Fiber Bioreactors (HFB, FiberCells Sys-
tem Inc., Frederick, MD) and then continuously cultured in 37 °C
and 5% CO
one fiber with 0.1
the HFB cartridge and polysulphone tubing, at a flow rate of
4 mL/min, bringing oxygen and nutrients to cells and removing
CO and other waste. We used collagen solution to create an extra-
2
. The HFB consists of a single, hydrophylic and polysulf-
lm diameter pores. The media circulate within
5
8%.
1
2
cellular matrix between cells and fiber. The polysulfone fiber was
coated with protein by flushing with 10 mL of coating solution
containing 1 mg collagen per 1 mL Phosphate Buffered Saline
2
-
.2.3. Preparation of N-[(7S)-1,2-dimethoxy-10-methylsulfanyl-9-oxo
3-(prop(2-en)oxy)-5,6,7,9-tetrahydrobenzo[ ]heptalen-7-yl]aceta
mide (3b) and N-[(7S)-3-ethoxy-1,2-dimethoxy-10-methylsulfanyl-9-
oxo-5,6,7,9-tetrahydrobenzo[ ]heptalen-7-yl]acetamide (3c)
a
(
PBS). In this manner CEM cells growing originally in suspension
a
build up a 3-D solid tumor. During 4 weeks of culturing, the media
were replaced each week.
One millimole of (3a) compound was dissolved in 2.5 mL of 1 M
sodium hydroxide solution. The resulting solution was cooled to
0
°C and 3-bromoprop-1-ene (1 mmol) for obtained compound
2
c)
.4. Preparation of media with colchicine derivatives (3a–c) and 4(a–
(
3b) or 1-bromoethane (1 mmol) for obtained (3c) was dissolved
in 3.5 mL acetone and added thereto. The solution was allowed
to stand for 15 h and then 25 mL of alkaline water was added.
Chloroform was used to extract the resulting product and drying
over magnesium sulfate. The yield of (3b) was 68% and (3c) was
The colchicine derivatives treated media were prepared using
nM, 10 nM, 20 nM, 100 nM, 500 nM and 1000 nM of (3a–c) and
1
(
4a–c) placed in a 1.5 mL glass vial and dissolved in 10 lL of di-
7
1%.
methyl sulfoxide. Dimethyl sulfoxide is a solvent of the (3a–c)
and (4a–c) derivatives. Once dissolved, the dimethyl sulfoxid/
2
.2.4. Preparation of the N-deacetyl-N-(N-trifluoroacetylaminoacyl)
(
3a–c) mixture or dimethyl sulfoxid/(4a–c) was added to media
thiocolchicine
and incubated overnight in 37 °C.
N-[(7S)-3-hydroxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-
5
,6,7,9-tetrahydrobenzo[
N-[(7S)-1,2-dimethoxy-10-methylsulfanyl-9-oxo-3-(prop(2-
en)oxy)-5,6,7,9-tetrahydrobenzo[ ]heptalen-7-yl]amine (4b);
N-[(7S)-3-ethoxy-1,2-dimethoxy-10-methylsulfanyl-9-oxo-
,6,7,9-tetrahydrobenzo[ ]heptalen-7-yl]amine (4c);
a
]heptalen-7-yl]amine (4a);
2.5. Preparation of media with colchicine derivatives (5a–c)
a
The media were supplemented with 1 nM, 10 nM, 20 nM,
100 nM, 500 nM and 1000 nM of (5a–c) derivatives dissolved in
10 lL of dimethyl sulfoxide. We observed that (5a–c) derivatives
5
a

D. Bartusik et al. / Bioorganic Chemistry 38 (2010) 1–6
3
were dissolved in media solution. However, we used dimethyl sulf-
oxide regime for the same media condition as for (3a–c) and (4a–
c) derivatives.
ACN water solution. A linear gradient from 5% to 70% ACN was ap-
plied over 60 min.
2.10. Antibody targeting of MHC (class I) receptor
2.6. Treatment of cells
The stock solution of 0.2 mg/mL antibodies in PBS pH 7.2 with
4
To establish IC50, 4 ꢀ 10 CEM cells/mL were treated on culture
plates and placed for the 72 h incubation with (3a–c), (4a–c) and
5a–c) derivatives. After 72 h, the growth was inhibited more than
0% for 20 nM of (3a), 10 nM of (3b) and (3c), 5 nM of (5a), (5b)
1
1
0 mg/mL bovine serum albumin (BSA) was used to treat
9
0 cell/mL.
(
5
2
.11. Statistical analysis
and (5c). Compounds (4a–c) were less active and we did not per-
form studies with (4a–c) in HFB device. Six synthesized com-
pounds (3a–c) and (5a–c) were tested in HFB device. Therefore,
we selected these compounds for 10 CEM cells/mL concentrations
for cells’ treatment in HFB, after 4 weeks in culture.
All results were expressed as a mean ± SD. Error bars in all
9
graphs represent the standard error of the mean. Differences
between groups at each time-point were identified by one-way An-
ova and the comparison between two independent variables was
determined by two-way Anova with Dunnet’s correction per-
formed post hoc to correct multiple comparisons. The p-values
2.7. Viability
<
0.05 were considered statistically significant. All data reported
The number of cells was determined manually with a hemacy-
tometer chamber (Hausser Scientific, Horsham, PA) using Trypan
blue (Sigma–Aldrich, Oakville, ON) exclusion method [12].
here are from sets of six separate experiments. The data was ana-
lyzed using the Sigma Stat Soft (Chicago, IL) software.
1
19
2
.8. Cell preparation for H and F Magnetic Resonance experiments
3. Results
1H and 19F MRI measurements of cells in the HFB were per-
Colchicine molecule (Fig. 1) is composed of three rings, a tri-
methoxy benzene ring, (ring A), a methoxy tropone ring (ring C),
and a seven-membered ring (ring B) carrying an acetamido group
at its C-7 position which anchors the A and C ring. Thiocolchicine
(2) was the starting compound for the preparation of a series of
thiocolchicine derivatives (3a–c), (4a–c) and (5a–c) (Fig. 2). Hydro-
lysis of acetamides (3a–c) with 20% of methanolic HCl gave the
amines (4a–c), respectively. The choice of introduction of the tri-
fluoroacetyl group at the C-7 resulted in (5a–c) compounds. These
functionalized N-fluoroacethylthiocolchicines were prepared from
(4a–c) compounds, respectively.
formed in control (n = 2, HFB) and treated cells (n = 6, HFB) using
nM, 5 nM, 10 nM, 20 nM, 100 nM, 500 nM and 1000 nM of (3a),
3b), (3c), (5a), (5b) and (5c) derivatives, respectively. Throughout
the MRI experiments, the HFBs were maintained under incubator-
like conditions (37 °C, 5% CO and 95% air). All MR images were
1
(
2
collected with 9.4 T (T)/21 cm magnets (Magnex, UK) and TMX
console (NRC-IBD, Canada). The HFBs with cell cultures were
placed in double tuned transmit/receive radio frequency (RF) vol-
ume coil operating at 376 MHz and 400 MHz corresponding to
1
1
9
9
1
1
F and H Larmour frequency at 9.4 T, respectively. All H and
F imaging parameters were the same for each HFB treated with
The control CEM cells cultured in the HFB reached density of
9
(
3a), (3b), (3c), (5a), (5b) and (5c) derivatives, respectively. The
10 cells/mL with the viability greater than 95% within 4 weeks.
MRI experiments were performed after 72 h of cell treatment in
Specifically, we used conventional culture CEM cells to establish
IC50 values. The growth inhibitory activity of thiocolchicine with
IC50 = 8.5 nM was about 5-fold lower than IC50 of colchicine
(40 nM). Considering thiocolchicine as the model compound, we
evaluated the effect of the substitution of 3-methyloxy or 7-acet-
amido group on the ring A or B in the thicolchicine derivatives ser-
ies on the CEM cell lines’ growth. Therefore, we determined the
IC50 values for all synthesized compounds. All thicolchicine deriv-
atives demonstrated strong cytotoxicity with mean IC50 values of
6.8 ± 3 nM. Compound (3c) with substitution at the C-3 position,
showed IC50 lower than IC50 of (3a–b). These results provided
motivation to use novel fluorine derivatives in CEM cell cultures.
We examined in vitro structures (5a–c) in the presence of fluorine
1
HFB device. For H MR imaging, a spin-echo pulse sequence was
used with Echo Time (TE)/Repetition Time (TR) = 16.5/5000 ms.
1
9
For F MR imaging, Inversion Recovery (IR) spin-echo method
with Inversion Time (IT) equal to 400 ms and TE/TR = 16.5/
5
000 ms were used. A single slice of 1 mm thickness was acquired
with matrix size of 256 ꢀ 256 and field of view 3 cm ꢀ 3 cm.
1
9
F spectra of pure (5a–c) solved in media were acquired using
one-pulse sequence (flip angle 60°; Repetition Time
TR) = 800 ms; number of average 2; Echo Time (TE) = 6 ms). The
concentration of each fluorinated compound in media was the
a
(
1
9
same as the one used to treat the cell culture. Moreover, F MRS
of culture cells treated with (5a–c) was performed using single
pulse measurements with parameters used to pure (5a–c) com-
pounds. Chemical shifts were referenced to an external standard
nuclei at the C-7 in the form of CF -group. What is interesting,
3
the IC50 values of (5a–c) were about 8-fold lower than IC50 of (2).
3
containing 1 lM CF COOH.
6
2
.9. High Performance Liquid Chromatography-Ultra Violet (HPLC-UV)
7
5
NHCOCH
3
analysis
B
7a
8
4a
Digested cell samples were fractionated with Gold HPLC chro-
matograph system equipped with Gold 166 Ultra Violet (UV)
Detector and 32-Karat software (Beckman-Coulter, Mississauga,
4
9
O
1a
1
2a
3
A
C
10
ON). For reversed-phase HPLC, a Vydac 218 TP54 Protein & Peptide
0
H
3
CO
12
C18 analytical column, 300 ÅA pore size, 0.46 cm ꢀ 25 cm (Separa-
2
1
11
OCH
3
tion Group, Hesperia, CA) was used. The chromatograph was
OCH
3
equipped with a Rheodyne injector (5 lL). UV detection was per-
formed at 245 nm. Eluent A consisted of 5% acetonitrile (ACN)
3
H CO
water solution and eluent B of 0.01% trifluoroacetic acid in 95%
Fig. 1. Structure of colchicine.

4
D. Bartusik et al. / Bioorganic Chemistry 38 (2010) 1–6
6
7
5
NHR
1
B
7a
8
4a
4
9
O
1a
1
2a
3
A
C
10
1
2
1
2
1
1
SCH
3
OCH
3
3
H CO
Fig. 2. Derivatives of thiocolchicine.
Thicolchicine derivatives with substitution at C-7 and C-3 showed
about 5-fold lower IC50 than thiocolchicine derivatives with substi-
tution at C-3. The IC50 for all of the nine compounds (3a–c), (4a–c)
and (5a–c) are presented in Fig. 3A–C. The data show that com-
pounds (3a–c) (1000 nM) and (5a–c) (1000 nM) induced about
4
9
0% growth inhibition of CEM cells at 4 ꢀ 10 cells/mL. Preferably,
the best kill effects in cell culture were provided by (3c) and (5b).
Compounds (4a–c) were less effective by inhibiting the growth of
7
0% of the same cell line at the same concentration. We did not
observe any decrease in cells’ growth for cells placed in 10
dimethyl sulfoxide only.
lL of
The results of the HPLC analysis of untreated and treated CEM
cells are shown in Fig. 4. The chromatograms showed expression
of MHC (class I) receptor eluted at 50–55 min in samples treated
with (3b) and (3c). Treatment with monoclonal antibody directed
to MHC (class I) receptor resulted in more than 90% killing effect.
Compounds (3b) and (3c) were more active in growth inhibition.
The HPLC fraction of the untreated cells contains only a major peak
at 5 min. Thus, the changes in profiles of treated cells correspond to
the changes in cells’ viability and cellular pathogenesis.
The use of 3-D cultured cells proved that CEM cells originally
cultured in suspension can form high density structure suitable
for MRI experiments. An important aspect of the current study
is that thiocolchicine derivatives suppressed cells number during
7
2 h of treatment and these changes are visible at Fig. 5B as
1
compared to initial tumor size at Fig. 5A. H MR image shows
cells’ aggregation in HFB before and after 72 h treatment. Be-
cause synthesized compounds (5a–c) have fluorine nuclei, we
1
9
used
F MRI to show changes in 3-D cells formation after
7
2 h. The 19F MR images (Fig. 5D) showed regions of cells with
19
fluorine derivative uptake. No F signal was observed in cell cul-
ture before treatment (Fig. 5C). The 19F MR image (Fig. 5D)
Fig. 3. Effects of various doses of derivatives on CEM cells. Data points indicated the
inhibitory percentage of cells ± SD in each treatment group: (3a–c) (A), (4a–c) (B)
and (5a–c) (C), as compared with controls.
showed regions of cells with fluorine derivative uptake. More-
1
9
over, no F signal was observed from extracellular media in
HFB device, because fresh media were flushed right after treat-
ment. Compounds (3a–c) and (5a–c) inhibited CEM cells growth
in HFB device with 90%. However, the IC50 were lower for (5a–c)
as presented in Table 1.
the chemical shift ꢁ10 ppm, ꢁ12 pm and ꢁ15 ppm, respectively.
The structure of each (5a–c) is based on colchicine, thus the chem-
ical shifts did not show differences larger than 5 ppm. In particular,
Fig. 6 shows the signal (ꢁ25 ppm) of the cells treated with (5c). As
The 19F MR spectra did not show any fluorine signal in cell cul-
ture before treatment. After treatment with (5a–c) fluorine signal
1
9
expected, the loss of pure 5c signal was visible on F MRS. The val-
1
9
was detectable by F MRS measurement in the treated cell cul-
tures. The chemical shifts of CF - in cell cultures were different
from pure (5a–c) compounds. The pure compounds (5a–c) have
3
ues of chemical shift of CF -group of (5a) and (5b) after treatment
1
9
3
were ꢁ20 ppm and ꢁ23 ppm. Each F MRS was repeated three
times.

D. Bartusik et al. / Bioorganic Chemistry 38 (2010) 1–6
5
Fig. 4. HPLC-UV chromatograms of untreated CEM cells (A), CEM cells treated with (3a) (B), CEM cells treated with (3b) (C), and CEM cells treated with (3c) (D).
3.1. Analytical analysis
4. Discussion
(
2) C22
H
25
N
1
O
5
S
1
; Calcd. for C22
H
25
N
1
O
5
S
1
: C, 63.60; H, 6.06; N,
Colchicine inhibits cancer cell growth by affecting mitose.
According to [13], the previous studies of large numbers of colchi-
cine analogues showed that derivatives modified at the B ring at C-
7 position can significantly influence the binding kinetics, associa-
tion rates, activation energy and the thermodynamics of the drug
binding to cells’ tubulin. Moreover, thiocolchicine available from
colchicine after treatment with sodium methanethiolate, is a po-
tent inhibitor of tubulin polymerization and cell growth, and binds
to tubulin more rapidly than colchicines [14]. In the literature,
there are examples of total suppression of animal tumors by col-
chicine in the Shope papilloma of the rabbit lymphosarcoma
[15], the mouse sarcoma [16] and the rat sarcoma [17]. The lack
of clinical interest in the colchicines seemingly arises from their
extreme toxicity. However, cancer cells are significantly more vul-
nerable to colchicine poisoning than normal cells. Therefore, many
attempts have been made to discover more effective and less toxic
analogues of colchicine by modifying basic structure of colchicine.
In our study, the effective concentrations of derivatives required to
induce the growth block in CEM cells were relatively low, in nM
range.
3
C
.37; S, 7.72. Found: C, 63.71; H, 6.15; N, 3.42; S, 7.79. (3a)
+
21
H
23
O
5
N
1
S
1
; requires M, 401.1. Found: EIMS m/e 401.1 (M );
: C, 62.8; H, 5.8; N, 3.5; S, 8.0. Found: C,
2.9; H, 5.8; N, 3.3; S, 7.5. (3b) C24 ; requires M, 454.5.
: C, 65.3; H,
.12; N, 3.17; S, 7.24. Found: C, 65.07; H, 6.59; N, 3.21; S, 7.28.
3c) requires M, 452.6. Found: EIMS 452.6
M Na ); Calcd. for C, 64.33; H, 18.64; N, 3.26; S, 7.45. Found: C,
23 5 1 1
Calcd. for C21H O N S
6
27 5 1 1
H O N S
+
+
27 5 1 1
Found: EIMS 454.5 (M Na ); Calcd. for C24H O N S
6
(
(
6
23 27 5 1 1
C H O N S ;
+
+
4.4; H, 18.9; N, 3.27, S% 7.61. (4a) C19
: C, 63.51; H, 5.91; N, 3.88; S, 8.92. Found: C, 63.55;
Calcd. for
: C, 65.8; H, 6.77; N, 3.52; S, 7.99. Found: C, 65.83;
H, 6.49; N, 3.63; S, 8.31. (4c) C21 ; C, 65.81; H, 6.50; N,
.6; S, 8.24. Found: C, 65.12; H, 6.54; N, 3.57; S, 8.27. (5a)
; Calcd. for C23 : C, 55.42; H, 4.61; N,
.2; S, 6.42; F, 11.44. Found: C, 55.43; H, 4.62; N, 2.91; S, 6.42; F,
1.44. (5b) C26 ; Calcd. for C26 : C, 56.52;
21 4 1 1
H O N S ; Calcd. for
C
19
21 4 1 1
H O N S
H, 5.83; N, 3.75; S, 8.93. (4b)
22 25 4 1 1
C H O N S ;
22 25 4 1 1
C H O N S
25 4 1 1
H O N S
3
C
2
1
23
H
23
O
6
N
2
S
1
F
3
23 6 2 1 3
H O N S F
H
27
O
6
N
S
2 1
F
3
27 6 2 1 3
H O N S F
H, 4.89; N, 5.07; S, 5.79; F, 10.32. Found: C, 56.52; H, 4.87; N,
.01; S, 5.79; F, 10.32. (5c)
7
C
25
H
27
O
6
N
2 1
S F
3
;
Calcd. for
C
25
H
27
O
6
N
2
S
1
F
3
: C, 57.03; H, 5.13; N, 5.32; S, 6.08; F, 10.87. Found:
The studies [18] on fluorine derivatives have reported promis-
ing effect of the trifluoroacetyl analogue on P388 leukemia in mice.
C, 53.67; H, 4.5; N, 5.32; S, 6.05; F, 10.85.

6
D. Bartusik et al. / Bioorganic Chemistry 38 (2010) 1–6
Fig. 6. 19F MRS of CEM cells treated with (5c). CF
reference at 0 ppm.
3
COOH was used as chemical shift
Fig. 5. An MR images of the CEM cells in the hollow fiber bioreactor at 9.4 T. The
solid red line indicates the area of cells and yellow solid line indicated the fiber. The
images: 1H MRI of cells before treatment with (5c) (A) and after 72 h treatment with
high enough. The results presented here show that MRI can iden-
tify suppressed regions of treated cells. Moreover, MRI can give a
unique insight into the treatment effects within a tumor over the
long course of treatment.
(
5c) derivative (B). Spin echo (SE) pulse sequence (TR/TE = 5000/12.8 ms,
19
FOV = 3 cm ꢀ 3 cm, slice thickness 1 mm and matrix 256 ꢀ 256) was used.
F
MRI of cells before treatment with (5c) (C) and after 72 h treatment with (5c)
1
9
derivative (D). The blue line at F images indicates contour of HFB device and the
green solid line indicated treated cells. Inversion Recovery (IR) spin-echo method
with Inversion Time (IT) equal to 400 ms and TE/TR = 16.5/5000 ms, slice thickness
5
. Conclusion
1
mm and matrix 256 ꢀ 256 was used.
In summary, the present study may explain some of the effects
of thiocolchicine derivatives on CEM cells. The following conclu-
sion on the structural changes can be drawn: thiocolchicine is
the active agent on the CEM cells’ growth inhibition; functionaliza-
tion of C-7 side chain with fluorine enhanced the growth inhibitory
activity of derivatives and the introduction of a trifluoromethyl
group in side chain of ring B increased the inhibitory activity of
derivatives ex vivo.
Table 1
IC50 values of the thiocolchicine derivatives in the HFB device measured with 109 CEM
cells/mL.
Derivatives
IC50 (nM)
(
(
(
(
(
(
3a)
3b)
3c)
5a)
5b)
5c)
5.0 ± 0.7
4.1 ± 1.1
4.2 ± 0.5
2.0 ± 0.8
1.5 ± 0.3
1.1 ± 0.6
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