The efficacy of new colchicine derivatives and viability of the T-Lymphoblastoid cells in three-dimensional culture using 19F MRI and HPLC-UV ex vivo

Article abstract of DOI:10.1016/j.bioorg.2009.07.007

The paper describes ex vivo applications of colchicine derivatives for the treatment of human T-Lymphoblastoid (CEM) cells. Moreover, the role of the substitutions of ring A at C-1 and C-7 side chain of colchicine analogues was probed by the synthesis and examination of their effects on the three-dimensional (3-D) CEM cells' growth. The CEM cells were cultured in the hollow fiber bioreactor (HFB) device. We used ¹H and ¹⁹F magnetic resonance imaging (MRI) to monitor changes in 3-D CEM cell culture. ¹⁹F MRI was used for visualization of the cellular uptake of new fluorine derivatives. Before and after treatment CEM cells profile was investigated with high performance liquid chromatography (HPLC-UV). Crown Copyright

Full text of DOI:10.1016/j.bioorg.2009.07.007

Bioorganic Chemistry 37 (2009) 193–201
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
The efficacy of new colchicine derivatives and viability of the T-Lymphoblastoid
cells in three-dimensional culture using 19F MRI and HPLC-UV ex vivo
Dorota Bartusik a,b, , Boguslaw Tomanek a,b,c,d, Erika Lattová e,f, Hélène Perreault e, Jack Tuszynski g,h
,
*
Gino Fallone b,d
a National Research Council Canada, Institute for Biodiagnostics (West), 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1
b Cross Cancer Institute, Medical Physics Department, 11560 University Ave, Edmonton, Alberta, Canada T6G 1Z2
c Polish Academy of Sciences, Institute of Nuclear Physics, Radzikowskiego 152, 31-342 Kraków, Poland
d University of Alberta, Oncology Department, 11560 University Ave, Edmonton, Alberta, Canada T6G 1Z2
e University of Manitoba, Department of Chemistry, 144 Dysart Road, Winnipeg, Manitoba, Canada R3T 2N2
f Slovak Academy of Sciences, Centre for Glycomics, 842 38 Bratislava, Slovakia
g Cross Cancer Institute, Experimental Oncology Department, 11560 University Ave, Edmonton, Alberta, Canada T6G 1Z2
h University of Alberta, Department of Physics, 11322 - 89 Avenue, Edmonton, Alberta, Canada T6G 2GZ
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 3 August 2009
The paper describes ex vivo applications of colchicine derivatives for the treatment of human T-Lympho-
blastoid (CEM) cells. Moreover, the role of the substitutions of ring A at C-1 and C-7 side chain of colchi-
cine analogues was probed by the synthesis and examination of their effects on the three-dimensional
(3-D) CEM cells’ growth. The CEM cells were cultured in the hollow fiber bioreactor (HFB) device. We
used 1H and 19F magnetic resonance imaging (MRI) to monitor changes in 3-D CEM cell culture. 19F
MRI was used for visualization of the cellular uptake of new fluorine derivatives. Before and after treat-
ment CEM cells profile was investigated with high performance liquid chromatography (HPLC-UV).
Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved.
Keywords:
Colchicine
Human T-Lymphoblastoid
Three-dimensional culture
19F Magnetic resonance imaging
High performance liquid chromatography
coupled with Ultra Violet
1. Introduction
with both colchicine and fluorinated colchicines derivatives using
1H and 19F MRI, respectively. While conventional 1H MRI detects
Colchicine (Fig. 1 (1)), has been widely used in immune-mediated
diseases [1,2]. The effects associated with the frequent occurrence of
drug resistance have prompted the search for new colchicine deriva-
tives. Moreover, recent studies have showed that colchicine inhibits
leukocyte-endothelial cell adhesion [3] and T-cells activation [4] by
bindingtointracellulartubulinmonomers,whichpreventstheirpoly-
merization[5].Thus,colchicinehasthepotentialtoimpairtheprocess
of antigen recognition and may inhibit the cancer cells’ growth.
In our experiments, the three-dimensional (3-D) cell cultures of
T-Lymphoblastoid (CEM) cells were treated with novel colchicine
and fluorinated colchicine derivatives. We monitored the cells’
growth in the Hollow Fiber Bioreactor (HFB) device using magnetic
resonance imaging (MRI), which enabled to visualize treatments
the signal from proton of mobile water in tissue, 19F MRI detects
intracellular uptake of fluorine containing drugs. The natural 19F
abundance is too low for 19F MRI detection, therefore the uptake of
fluorine derivatives of colchicine in cells can be observed without
any additional contrast agents.
Several studies have shown that colchicine derivatives with
functionalized side chain at C-7 position are promising for treat-
ment of lymphocytic leukemia [6,7]. Therefore we synthesized
fluorinated derivatives of colchicine with the modifications in posi-
tions C-1 and C-7. Moreover, MRI technique used in this study was
suitable for multiple, repeated measurements to observe dynamic
changes in response to treatment and provided noninvasive ex vivo
characteristics of the 3-D tumor.
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, mag-
netic resonance imaging.
* 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.
2. Materials and methods
2.1. Chemicals and reagents
Colchicine, N-[(7S)-1,2,3,10-tetramethoxy-9-oxo-5,6,7,9-tetra-
E-mail address: Dorota.Bartusik@gmail.com (D. Bartusik).
hydrobenzo[a]heptalen-7-yl] acetamide and all chemical com-
0045-2068/$ - see front matter Crown Copyright Ó 2009 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.bioorg.2009.07.007

194
D. Bartusik et al. / Bioorganic Chemistry 37 (2009) 193–201
pounds used to the synthesis of colchicines derivatives were pur-
chased from Sigma–Aldrich (Oakville, ON).
tracted with 10 mL of CH2Cl2 and washed with brine. The ex-
tracts were dried over Na2SO4 and concentrated to 3 mL. The
deacetylated compounds (17–27) were crystallized from 10 mL
of CH2Cl2.
2.2. Synthesis
One millimole of deacetylated compound (17–27) and N-tri-
fluoroacetylamino acid (1 mmol) was dissolved at room temper-
ature in CH2Cl2 (6 mL). N-trifluoroacetylamino acid was N-trifluo-
roacetylglycine that was prepared from glycine by protection at
the nitrogen atom with the trifluoroacetyl group [9]. As condens-
ing compound 0.5 mmol of dicyclohexylcarbodiimide (DCC) was
used. After 2 h, the suspension was cooled to 0 °C and filtrated.
Products (28–38) were chromatographed on silica gel column
eluting with dichloromethane/methanol (1:0–0:1). Crystallization
of (28–38) were performed with dichloromethane: ethyl ether
(1:1).
2.2.1. Preparation of N-[(7S)-2,3,10-trimethoxy-1-((methyl)carbonyl-
oxy)-9-oxo-5,6,7,9-tetrahydrobenzo[
and N-[(7S)-1-hydroxy-2,3,10-trimethoxy-9-oxo-5,6,7,9-tetrahydro-
benzo[ ]heptalen-7-yl]acetamide (3)
a]heptalen-7-yl]acetamide (2)
a
Compounds (2) and (3) were obtained by a method described
by Pontakis et al. [8], as shown in Fig. 1.
2.2.2. Preparation of N-[(7S)-1-((ethyl)carbonyloxy)-2,3,10-trimeth-
oxy-9-oxo-5,6,7,9-tetrahydrobenzo[
and N-[(7S)-1-(((methyl)ethyl)carbonyloxy)-2,3,10-trimethoxy-9-
oxo-5,6,7,9-tetrahydrobenzo[ ]heptalen-7-yl]acetamide (5)
a]heptalen-7-yl]acetamide (4)
a
1 mmolof derivative (3) was dissolvedindry pyridine and 1 mmol
of propionyl chloride for preparation of (4) or isobutyryl chloride for
preparation of (5) was added at 0 °C. The solution was allowed to
stand overnight and then diluted with 10 mL of water and extracted
with ethyl acetate (2 ꢀ 10 mL). The organic layer was washed with
5 mL of brine, dried with Na2SO4 and concentrated to 3 mL. Com-
pounds (4) and (5) presented in Fig. 2 were crystallized from CH2Cl2.
2.3. Cell cultures
CEM cells (American Type Culture Collection, Manassas, VA)
were maintained in tissue culture flasks and cultured as monolayer
in 20 mL of RPMI media containing 10% Fetal Bovine Serum (FBS).
When the number of cells in the culture flask reached 5–
6 ꢀ 106 cells/mL, the culture was harvested and then inoculated
into fifteen Hollow Fiber Bioreactors (HFB, Fiber Cells System
Inc., Frederick, MD) and continuously cultured in 37 °C and 5%
CO2. The media circulating within the HFB cartridge and polysul-
phone tubing, at flow rate of 14 mL/min, bring oxygen and nutri-
ents to cells and remove CO2 and other waste. HFB device with
1 cm diameter consists of a single, hydrophylic and polysulfone
2.2.3. Preparation of N-[(7S)-1-(ethoxy)-2,3,10-trimethoxy-9-oxo-
5,6,7,9-tetrahydrobenzo[
((ethoxy)-1-methyl-2,3,10-trimethoxy-9-oxo-5,6,7,9-tetrahydroben-
zo[ ]heptalen-7-yl] acetamide (7); N-[(7S)- 2,3,10-trimethoxy-9-oxo-
1-(propanoxy)-5,6,7,9-tetrahydrobenzo[ ]heptalen-7-yl]acetamide
(8); N-[(7S)-2,3,10-trimethoxy-9-oxo-1-((prop(2-en)oxy)-5,6,7,9-tet-
rahydrobenzo[ ]heptalen-7-yl]acetamide (9); N-[(7S)-2,3,10-trimeth-
oxy-9-oxo-1-((phenyl)methoxy)-5,6,7,9-tetrahydrobenzo[ ]heptalen-
7-yl]acetamide (10); N-[(7S)-2,3,10-trimethoxy-9-oxo-1-(((3-metho-
xy)propane)oxy)(3-methoxy))- 5,6,7,9-tetrahydrobenzo[ ]heptalen-
7-yl]acetamide (11); N-[(7S)-2,3,10-trimethoxy- 9-oxo-1-((phenyl(3-
chloro))methoxy)-5,6,7,9-tetrahydrobenzo[ ]heptalen-7-yl]acetamide
(12); N-[(7S)-2,3,10-trimethoxy- 9-oxo-1-((pyridine(3))yl)-5,6,7,9-te-
trahydrobenzo[ ]heptalen-7-yl]acetamide (13); N-[(7S)-2,3,10-trime-
thoxy- 9-oxo-1-((phenyl(2-chloro))methoxy)-5,6,7,9-tetrahydroben-
zo[ ]heptalen-7-yl] acetamide (14); N-[(7S)-2,3,10-trimethoxy- 9-ox-
o-1-(((phenyl(4-chloro))methoxy)-5,6,7,9-tetrahydrobenzo[ ]heptal-
en-7-yl] acetamide (15); N-[(7S)-2,3,10-trimethoxy-1-((methyl)cyclo-
a]heptalen-7-yl] acetamide (6); N-[(7S)-1-
a
a
a
fiber with 0.1 lm diameter pores. We used collagen solution to
a
create an extracellular matrix between the cells and the fiber.
The polysulphone fiber was coated with protein by flushing with
10 mL of coating solution containing 1 mg of collagen per 1 mL
Phosphate Buffered Saline (PBS). Due to perfusion, the HFB ab-
sorbed sufficient oxygen from the reservoir with fresh media to
keep cells alive. The perfusion medium was changed weekly when
the glucose level reached 2 g/L measured with a glucometer. The
number of cells was determined manually with a hemacytometer
chamber (Hausser Scientific, Horsham, PA) using Trypan blue
exclusion method [10].
a
a
a
a
a
hexane)- 9-oxo-5,6,7,9-tetrahydrobenzo[
(16)
a]heptalen-7-yl] acetamide
2.4. Cell treatment
One millimole of derivative (3) was dissolved in 2.5 mL of so-
dium hydroxide solution and was cooled to 0 °C. 1 mmol of bro-
mide derivatives (e.g. 1-bromoethane for preparation of (6), 2-
methylo-1-bromoethane for (7), 1-bromopropane for (8), 3-
bromoprop-1-ene for (9), ((bromo)methyl)benzene for (10), 1-
methoxy-2-bromoethene for (11), 1-bromo-3-chlorobenzene for
(12), 3-((bromo)methyl)pyridine for (13), 1-bromo-1-chloroben-
zene for (14), 1-bromo-4-chlorobenzene for (15), 1-((bromo)-
methyl) cyclohexane for (16)) was dissolved in 3.5 mL acetone
and each solution was allowed to stand for 15 h, then 25 mL of
alkaline water was added. Chloroform (2 ꢀ 10 mL) was used to ex-
tract the compounds, while washing was effected with 15 mL of
water. The organic layers were dried with Na2SO4 and concen-
trated to 3 ml. The compounds (6–16) were crystallized from
10 mL of CH2Cl2. The syntheses of (6–16) are presented in Fig. 3.
To establish IC50, 4 ꢀ 104 CEM cells/mL in six well microplates
were used to determine the activity of (1–38) compounds. Solu-
tions of the treated media with (1–38) were prepared using:
1 nM, 10 nM, 20 nM, 100 nM, 500 nM and 1000 nM of (1–38)
placed in a 1.5 mL glass vials and dissolved in 10
lL of dimethyl
sulfoxide. 10 L of dimethyl sulfoxide was the solvent for each
l
(1–38) derivative. Once dissolved, the dimethyl sulfoxid/(1–38)
appropriate mixtures were added to the media and incubated
overnight in 37 °C. The cells were exposured to (1–38) and incu-
bated for 72 h. After treatment, 0.4% (w/v) Trypan blue dye solu-
tion was added and cell viability was then determined using a
hemacytometer chamber. Moreover, CEM cells were treated with
10
examined.
To treat 3-D cultures in the HFB device for 72 h we selected (6),
lL of dimethyl sulfoxide alone and viability after 72 h was also
2.2.4. General procedure for the preparation of N-deacetyl-N-(N-
trifluoroacetylaminoacyl) colchicine
Three millimoles of appropriate derivative (6–16) dissolved in
methanol (50 mL) and 2 M HCl (25 mL) were heated at 90 °C with
simultaneous stirring for 1 day. Next, the reaction mixture was
cooled and neutralized with NaHCO3. Products (17–27) were ex-
(13), (28) and (35) compounds, because for these colchicine deriv-
atives the cells’ growth was inhibited over 50% for relatively low
concentration of 20 nM. For each treatment with 1000 nM of (6),
(13), (28) and (35) three HFBs were used (n = 12 for treatment;
n = 3 for control). Viability of cells removed from the HFB was mea-
sured with Trypan blue.

D. Bartusik et al. / Bioorganic Chemistry 37 (2009) 193–201
195
6
6
7
7
5
NHCOCH3
7a
5
NHCOCH3
7a
SnCl4
8
B
8
4a
B
4
4a
4
CH3COCl
9
O
9
1a
O
1a
12a
3
C
12a
A
3
C
A
10
10
12
H3CO
H3CO
12
H3CO
1
2
1
OCH311
2
OCH3
11
OCH3
OCCH3
H3CO
(1)
(2)
O
6
7
5
NHCOCH3
7a
8
K2CO3
B
1a
4a
4
9
O
12a
3
C
A
10
12
H3CO
H3CO
1
2
11
OCH3
OH
(3)
Fig. 1. Structures of the compounds (1), (2) and (3).
2.5. MRI
19F MR imaging was performed to determine the intracellular
19F SI of (28) and (35) on the cell cultures in HFBs devices. To opti-
mize the intracellular uptake of (28) and (35) the phantoms con-
sisting of HFB tubes filled with 103 cells, 104 cells, 105 cells, 106
cells, 107 cells, 108 cells and 109 cells and 1 nM, 10 nM, 20 nM,
100 nM, 500 nM and 1000 nM of (28) or (35) were used. It is al-
ready known, that 19F SI is linearly related with the amount of
19F molecules taken up by cells [11]. Therefore, the linear regres-
sion was used to find the 19F SI dependence on treated cell num-
bers. The 19F SI measured from axial slices of tubes filled with
pure (28) and (35) without cells were considered as 100% of SI. Per-
centage change in SI (19F SI (% change)) was normalized to the SI of
pure (28) and (35) with the use of the following equation: 19F SI (%
change) = [(U ꢁ L)/U] ꢀ 100%, where (U) was pure (28) and (35)
and L = 19F SI of (28) or (35) in each samples containing different
cell numbers. To measure intracellular uptake of drug without
19F background from treated media, the cells in HFB device after
All MRI experiments were performed using a 9.4 Tesla (T) with
21 cm bore magnet (Magnex, UK) and TMX console (NRC-IBD, Can-
ada). Images were acquired using double (19F and 1H) tuned trans-
mit/receive Radio Frequency (RF) volume coil operating at
376 MHz and 400 MHz corresponding to 19F and 1H Larmour fre-
quency at 9.4 T, respectively. For 1H MRI, a spin echo pulse se-
quence was used with Echo Time (TE)/Repetition Time
(TR) = 16.5/5000 ms. 19F MRI was performed with Inversion Recov-
ery (IR) spin echo method with Inversion Time (IT) of 400 ms and
TE/TR = 16.5/5000 ms. For imaging of HFBs, axial slices with 1 mm
thickness were defined perpendicular to the HFB’s long axis and
field of view was set to 3 ꢀ 3 cm, matrix size of 256 ꢀ 256 resulting
in a resolution of 117 ꢀ 117
l
m for 1H and 19F MRI. 1H MR images
were collected before drug introduction and after 72 h of exposure
to (6), (13), (28) and (35).
6
7
6
5
NHCOCH3
7
5
NHCOCH3
8
7a
B
1a
RCOCl
4a
4
8
9
7a
B
1a
4a
4
O
9
O
12a
C
3
A
12a
C
3
A
10
12
10
H3CO
H3CO
OCH3
1
12
2
11
1
2
11
OH
OCH3
OCR
H3CO
H3CO
(3)
(4, 5)
O
(4) R = CH3CH2
(5) R = (CH3)2CH
Fig. 2. Scheme of the preparation of (4–5).

196
D. Bartusik et al. / Bioorganic Chemistry 37 (2009) 193–201
6
7
6
5
R2Br
NHR1
7
NHR1
5
8
B
7a
4a
4
9
8
7a
B
O
4
4a
9
1a
O
12a
1a
C
3
A
12a
C
3
A
10
12
10
H3CO
1
H3CO
12
2
11
OCH3
2
11
1
OH
OCH3
O
R2
H3CO
H3CO
(3)
(6-16)
6
6
7
7
5
NHR1
CH2 (COOH)NH(CO)CF3
5
NHR3
8
7a
B
1a
12a
12
4a
8
7a
B
4
4a
4
9
9
O
O
1a
C
3
12a
A
C
3
A
10
10
H3CO
12
H3CO
OCH3
1
2
11
1
2
11
OCH3
O
R2
(17-27)
O
R2
(28-38)
H3CO
H3CO
(6) R2 – CH2CH3; R1 – (CO)CH3
(7) R2 – CH(CH3)2; R1 – (CO)CH3
(17) R2 – CH2CH3; R1 – H
(28) R2 – CH2CH3; R3 – (CO)CH2NH(CO)CF3
(29) R2 – CH(CH3)2; R3 – (CO)CH2NH(CO)CF3
(30) R2 – CH2CH2CH3; R3 – (CO)CH2NH(CO)CF3
(31) R2 – CH2CH=CH2; R3 – (CO)CH2NH(CO)CF3
(32) R2 – CH2(C6H5); R3 – (CO)CH2NH(CO)CF3
(33) R2 – CH2CH2OCH3; R3 – (CO)CH2NH(CO)CF3
(34) R2 – CH2(C6H4)-m-Cl; R3 – (CO)CH2NH(CO)CF3
(35) R2 – CH2(ϕ-C5H4N); R3 – (CO)CH2NH(CO)CF3
(36) R2 – CH2(C6H4)-o-Cl; R3 – (CO)CH2NH(CO)CF3
(37) R2 – CH2(C6H4)-p-Cl; R3 – (CO)CH2NH(CO)CF3
(38) R2 – CH2(C6H11); R3 – (CO)CH2NH(CO)CF3
(18) R2 – CH(CH3)2; R1 – H
(19) R2 – CH2CH2CH3; R1 – H
(20) R2 – CH2CH=CH2; R1 – H
(21) R2 – CH2(C6H5); R1 – H
(8) R2 – CH2CH2CH3; R1 – (CO)CH3
(9) R2 – CH2CH=CH2; R1– (CO)CH3
(10) R2 – CH2(C6H5); R1 – (CO)CH3
(11) R2 – CH2CH2OCH3; R1 – (CO)CH3
(12) R2 – CH2(C6H4)-m-Cl; R1 – (CO)CH3
(13) R2 – CH2(ϕ-C5H4N); R1– (CO)CH3
(14) R2 – CH2(C6H4)-o-Cl; R1– (CO)CH3
(15) R2 – CH2(C6H4)-p-Cl; R1 – (CO)CH3
(16) R2 – CH2(C6H11); R1– (CO)CH3
(22) R2 – CH2CH2OCH3; R1 – H
(23) R2 – CH2(C6H4)-m-Cl; R1 – H
(24) R2 – CH2(ϕ-C5H4N); R1 – H
(25) R2 – CH2(C6H4)-o-Cl; R1 – H
(26) R2 – CH2(C6H4)-p-Cl; R1 – H
(27) R2 – CH2(C6H11); R1 – H
Fig. 3. Scheme of the syntheses (6–38).
treatment were washed with fresh media. The media were pump-
ing with flow rate of 14 mL/min and 5 mL sample from media res-
ervoir was taken after every washing to check 19F SI. The washing
of cells was repeated until 19F SI of media was to low for measure-
ments with 19F MRI at 9.4 T. Data analysis was proceeded with
post-processing software MAREVISI (NRC-IBD, Canada).
formed at 245 nm. Eluent A consisted of 5% acetonitrile (ACN)
water solution and eluent B of 0.01% trifluoroacetic acid in 95%
ACN water solution. A linear gradient from 5% to 70% ACN was ap-
plied over 60 min.
2.7. Statistical analysis
2.6. HPLC-UV
Results were expressed as a mean SD. Differences between
groups at each time-point were identified by one-way Anova. Sta-
tistical comparison between two independent variables was deter-
mined by two-way Anova with Dunnet’s correction performed post
hoc to correct multiple comparisons. The p-values <0.05 were con-
sidered statistically significant. All data reported here are from sets
of three separate experiments. Error bars in all graphs represent
the standard error of the mean. Data were analyzed using the Sig-
ma Stat Soft software (Chicago, IL).
Digested cell samples were fractionated with a Gold HPLC chro-
matograph system equipped with a Gold 166 Ultra Violet (UV)
Detector and 32-Karat software (Beckman-Coulter, Mississauga,
ON). For reversed-phase HPLC, a Vydac 218 TP54 Protein & Peptide
0
C18 analytical column, 300 ÅA pore size, 0.46 cm ꢀ 25 cm (Separa-
tion Group, Hesperia, CA) was used. The chromatograph was
equipped with a Rheodyne injector (5 lL). UV detection was per-

D. Bartusik et al. / Bioorganic Chemistry 37 (2009) 193–201
197
respectively. The viable cells are visible with 1H MRI and their
number decreased during time exposure to the drugs, which corre-
sponds to decrease of cells volume observed on the 1H image. The
data indicate that the regions of cells with low signal intensities
(SI) (more darker) are the regions with more dead cells, as exam-
ined with Trypan blue. The loss of intracellular water in region of
dead cells confirmed the differences in SI. The total region of cells
visible with 1H MRI decreased for 25 3% and 35 6% as compared
to regions before treatment with (6) and (13), respectively. These
data imply that the flow of medium through the HFB center pro-
vides a nutrient source and clearance of waste products and that
the cells are with high densities close to the fiber. Accordingly,
the region of cells with high densities before treatment, possesses
more viable cells after treatment. Particularly, the number of cells
before and after treatment proved that close to the fiber the den-
sity of cells was 10% higher as compared to others region of HFB.
19F MR images were collected in treated cells at the same imag-
ing session and the same geometry that were used to 1H MR
images. The treatment effect on 19F MR images was the increased
19F SI due to increased cellular uptake of fluorine drug and dead
cells, therefore 19F SI was measured for the cells treated in HFB de-
vice with fluorine derivatives (28) and (35). Following previously
published recommendation, 19F SI was measured directly from ax-
ial slice of HFB device [12]. Moreover, 19F MRI selectively visualizes
only intracellular fluorine uptake with no background and shows
distribution of derivatives in cell cultures. As proved by viability
assay, the cells visible with 19F MRI are nonviable cells while exam-
ined by Trypan blue. The distribution of cells can be measured by
MRI and in the HFB device, the cells possessed density distribution.
The significantly higher number of cells was killed in the regions
where cells’ density was high. Fig. 6C and D are 19F images and
showed uptake of (28) and (35) derivatives of colchicines in dead
cells. No 19F signal was observed from extracellular media, for in-
stance compounds that have not been taken up by cells because
fresh media was flushed right after treatment. SI was measured
as a regional signal and then the mean value was calculated.
The data presented in Figs. 7 and 8 illustrated the optimization
process of ex vivo applications of (28) and (35). The 19F SI was
linear with respect to 19F content in known numbers of cells.
Moreover, both figures, Figs. 7 and 8, illustrated that: the concen-
tration of emulsions can be quantified based on the 19F SI and the
number of targeted cells can be counted based on 19F SI values.
Therefore, we estimated the number of CEM cells labeled with
19F-derivatives of colchicines in HFB devices. Study showed that,
within 72 h cells exposure to 1000 nM of (28) and (35), the num-
3. Results
Colchicine analogues synthesized and tested for their ability to
inhibit CEM cell growth ex vivo were separated into three groups
and are presented in Figs. 1–3. The syntheses of the colchicine
derivatives started with the conversion of known classical colchi-
cine structure to ester or ether structure at the position C-1. The
hydrolysis of (2) in the presence of K2CO3 produced compound
(3) (Fig. 1) with 71% yield. The esterification of (3) afforded ester
derivatives (4–5) as shown in Fig. 2. The class of compounds (6–
16) were obtained by etherification of (3) (Fig. 3). Among these
compounds, fluorine derivatives (28–38) were synthesized using
(17–27) as started compounds (Fig. 3).
The 3-D cell cultures in the HFB device reached 109 CEM cells/
mL after 4 weeks. The 72 h incubations of cells with (6–16) and
(28–35) decreased cells viability and showed the ability of the ana-
logues to accumulate and interact within cells. The observed IC50 of
the cells’ growth inhibition using colchicine analogues are summa-
rized in Fig. 4. The analogues of (6–16) exhibited a similar effect
with main value IC50 = 13 1 nM. However, (28–35) analogues
showed
a
higher decreases in cells viability and main
IC50 = 7 2 nM. The observed IC50 suggested a lower capacity to
antagonize cells’ growth for (6–16) derivatives than (28–35). The
fluorinated analogues (28–35) were the most effective compounds
in all studied derivatives (1–38). After the preliminary test of (17–
27) we did not perform viability studies using these derivatives,
because the observed effects did not show decreases of cells’ viabil-
ity higher than the precursor (1). However, the compounds (6) and
(13) showed significant changes in IC50 values. Based on these re-
sults, we selected (6) and (13) as well as their fluorinated ana-
logues (28) and (35) for the studies in HFB device. The influence
of the investigated compounds on 3-D CEM cell growth was con-
firmed with cell viability binding assays. As shown in Fig. 5, the
compounds (6) or (13) and fluorinated analogues (28) or (35) were
able to induce high growth inhibition effect in HFB cultures. Viabil-
ity of the control (untreated) cells during culture was 93 2%. Cells
treated with 10
lL of dimethyl sulfoxide did not show any de-
creases in growth.
To observe the 3-D cells aggregation in the HFB device, the cells
before and after treatment with (6), (13), (28) and (35) were im-
aged with the use of 1H MRI. The 1H images allowed to monitor
the localization of cells around the fiber device, thus providing ana-
tomical images of the cultures. Fig. 6A and Fig. 6B are 1H images
with the resolution of 117 ꢀ 117
lm and present the cross-section
of the entire HFB device with cells treated with (6) and (13),
Fig. 4. IC50 values of (1–38).

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D. Bartusik et al. / Bioorganic Chemistry 37 (2009) 193–201
Fig. 5. Viability of cells treated with (6), (13), (28) and (35).
Fig. 6. 1H MRI of CEM cells treated with (6) (A); 1H MRI of cells treated with (13) (B); 19F MRI of cells treated with (28) (C); 19F MRI of cells treated with (35) (D). The yellow
solid line indicated the cells region (A–D), the blue solid line indicated the fiber (A, B) and the green solid line indicated region with higher uptake of fluorine derivatives (C, D).
The scale of (A–D) to original image is 3:1.
bers of viable cells decreased from 109 cells/mL to 3.45 ꢀ
108 cells/mL and from 109 cells/mL to 2.9 ꢀ 108 cells/mL, respec-
tively. The mean 19F SI of the cells treated with (28) increased
during treatment and corresponding to a mean cells concentra-
tion of 6.03 ꢀ 108 cells/mL. The mean CEM cell density in the
region with lower densities corresponds to 2.4 ꢀ 108 cells/mL
while the mean numbers of cells with higher cells densities cor-
responds to 3.5 ꢀ 108 cells/mL. At the same time the viability of
cells in HFB treated with (28) was 35% and corresponding to
3.45 ꢀ 108 cells/mL viable cells in HFB.
The mean 19F SI of the cells treated with (35) also increased dur-
ing treatment and corresponded to a mean cells concentration of
6.9 ꢀ 108 cells/mL. The viability of cells in HFB treated with (35)
was 30% and corresponded to live cells 2.9 ꢀ 108 cells/mL after
3 days of treatment. The mean CEM cell density in the region with
lower densities corresponded to 1.4 ꢀ 108 cells/mL while the mean
numbers of cells with higher cells densities were 4.8 ꢀ 108 cells/
mL. As expected, the 19F SI values were dependent on the concen-
tration of cells and fluorine derivatives in the cells treated with
(28) (Fig. 7) and (35) (Fig. 8).

D. Bartusik et al. / Bioorganic Chemistry 37 (2009) 193–201
199
Fig. 7. Number of cells vs. increase of 19F SI for (28) (100% corresponds to SI of pure (28) without cells).
Moreover, we found, that 3-D high density cell cultures were
needed for the study, due to the limited MRI sensitivity. We also
found, that the HFB provides high enough concentration of cells
to obtain 1H and 19F MR images, thus the combined MRI techniques
and HFB device can be used for studying fluorine drug efficacy and
cells viability.
The results of the HPLC analysis of the treated CEM cells
ex vivo are shown in Fig. 9A–E. Particularly, Fig. 9A showed frac-
tion of untreated CEM cells. As shown in Fig. 9C and E, the cells in
response to treatment with (13) and (35) expressed the minor
histocompatibility complex MHC (class I) receptor eluted at
52 min. When the viability was 45% and 35%, the expression of
MHC (class I) receptor was observed with intensities of 0.05 Volt
(V) and 0.7 V, respectively. The exposure of cells to (6), (13) and
(28) showed a new HPLC peak eluted at 23 min with low inten-
sity, the Tn receptor (Fig. 9B–D). The signal of Tn in cells treated
with (13) had intensity 10 times higher (Fig. 9C) in 45% viable cell
culture than treated with (6) in 50% viable cell culture (Fig. 9B)
and with (28) in 38% viable cell culture (Fig. 9D). We assumed
that signals eluted at 30–35 min were unreacted derivatives with
variable intensity of 0.1 V for (28) and (35) as well as 0.35 V for
(13) and 0.3 V for (6). The viability of cell cultures were higher
for samples where unreacted colchicine derivatives were pre-
sented with higher intensities and were as follow: 38% 4 for
(28), 35% 5 for (35), 45% 2 for (13) and 50% 4 for (6). Addi-
tional signal from cascade of apoptotic proteins was eluted at
56 min and occurred in samples treated with (28) and (35)
(Fig. 9D and E). The peaks at 56 min with intensity of 0.35 V
(28) and 0.2 V (35) were measured in cells with viability of 38%
(28) and 35% (35). The undefined additional peaks with very
low intensities, less than 0.05 V, are the metabolites of derivatives
or unreacted compounds.
Fig. 8. Number of cells vs. increase of 19F SI for (35) (100% corresponds to SI of pure (35) without cells).

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D. Bartusik et al. / Bioorganic Chemistry 37 (2009) 193–201
Fig. 9. HPLC chromatograms of untreated CEM cells (A); cells treated with (6) (B); cells treated with (13) (C); cells treated with (28) (D) and cells treated with (35) (E).
3.1. Analytical analysis
C28H28O6N1Cl1; requires M, 509. Found EIMS m/e 509.1 (M+);
Calcd. for C28H28O6N1Cl1: C, 66.01; H, 5.50; N, 2.94; Cl, 6.87. Found:
C, 66.03; H, 5.51; N, 2.95; Cl, 6.88. (15) C28H28O6N1Cl1; requires M,
509. Found EIMS m/e 509.1 (M+); Anal. Calcd. for C28H28O6N1Cl1: C,
65.01; H, 6.09; N, 3.16; Cl, 7.90. Found: C, 65.02; H, 6.07; N, 3.10;
Cl, 7.92. (16) C28H35O6N1; requires M, 481. Found EIMS m/e 481.2
(M+); Calcd. for C28H35O6N1: C, 69.85; H, 7.27; N, 2.91. Found: C,
70.04; H, 7.08; N, 2.93. (17) C21H25O5N1; Anal. Calcd. for
C21H25O5N1: C, 67.92; H, 7.27; N, 3.77. Found: C, 67.93; H, 7.28;
N, 3.78. (18) C22H27O5N1 Calcd. for C22H27O5N1: C, 68.57; H, 7.01;
N, 3.77. Found: C, 68.59; H, 7.03; N, 3.79. (19) C22H27O5N1; Calcd.
for C22H27O5N1: C, 68.57; H, 7.01; N, 3.78. Found: C, 68.62; H,
7.05; N, 3.79. (20) C22H25O5N1; Calcd. for C22H25O5N1: C, 68.92;
H, 6.52; N, 3.65. Found: C, 68.94; H, 6.53; N, 3.67. (21)
C26H27O5N1; Calcd. for C26H27O5N1: C, 72.05; H, 6.23; N, 3.23.
Found: C, 72.21; H, 6.04; N, 3.23. (22) C22H27O6N1; Calcld. for
C22H27O6N1: C, 65.83; H, 6.73; N, 3.49. Found: C, 65.82; H, 6.73;
N, 3.48. (23) C26H26O5N1Cl1; Calcd. for C26H26O5N1Cl1: C, 66.80;
H, 5.56; N, 2.99; Cl, 7.49. Found: C, 66.93; H, 5.34; N, 3.01; Cl,
7.53. (24) C22H26O5N1; Calcd. for C22H26O5N1: C, 68.75; H, 6.77;
N, 3.64. Found: C, 81.26; H, 6.78; N, 3.66. (25) C26H26O5N1Cl1;
Calcd. for C26H26O5N1Cl1: C, 66.80; H, 5.56; N, 2.99; Cl, 7.49. Found:
C, 66.81; H, 5.55; N, 2.98; Cl, 7.48. (26) C26H26O5N1Cl1; Calcd. for
C26H26O5N1Cl1: C, 66.80; H, 5.56; N, 2.99; Cl, 7.49. Found: C,
66.81; H, 5.57; N, 2.85; Cl, 7.51. (27) C26H33O5N1; Calcd. for
C26H33O5N1: C, 76.53; H, 7.51; N, 3.18. Found: C, 76.22; H, 7.32;
N, 3.20. (28) C25H27O7N2F3; Calcd. for C25H27O7N2F3: C, 57.25; H,
5.15; N, 5.18; F, 10.85. Found: C, 57.25; H, 4.99; N, 5.34; F, 10.86.
(29) C26H29O7N2F3; Calcd. for C26H29O7N2F3: C, 57.99; H, 5.39; N,
5.20; F, 10.59. Found: C, 56.38; H, 5.3; N, 5.3; F, 10.87. (30)
(2) C23H25O7N1; requires M, 427. Found EIMS m/e 427.1 (M+);
Calcd. for C23H25O7N1: C, 64.63; H, 5.85; N, 3.27. Found: C, 64.64;
H, 5.86; N, 3.31. (3) C21H23O6N1; requires M, 385. Found EIMS m/
e 385.1 (M+); Calcd. for C21H23O6N1: C, 65.45; H, 5.97; N, 3.63.
Found: C, 65.43; H, 5.99; N, 3.71. (4) C24H27O7N1; requires M,
441. Found EIMS m/e 441.1 (M+); Calcd. for C24H27O7N1: C,
65.30; H, 6.12; N, 3.72. Found: C, 65.31; H, 6.12; N, 3.72. (5)
C25H29O7N1; requires M, 455. Found EIMS m/e 455.0 (M+); Calcd.
for C25H29O7N1: C, 65.93; H, 6.37; N, 3.07. Found: C, 65.94; H,
6.38; N, 3.08. (6) C23H27O6N1; requires M, 413. Found EIMS m/e
413.1 (M+); Calcd. for C23H27O6N1: C, 66.83; H, 6.55; N, 23.22.
Found: C, 66.82; H, 6.54; N, 23.22. (7) C24H29O6N1; requires M,
427. Found EIMS m/e 427.1 (M+); Calcd. for C24H29O6N1: C,
67.44; H, 6.77; N, 3.22. Found: C, 67.41; H, 6.73; N, 3.21. (8)
C24H29O6N1; requires M, 427. Found EIMS m/e 427.1 (M+); Calcd.
for C24H29O6N1: C, 67.44; H, 6.79; N, 32.78; Found: C, 67.44; H,
6.80; N, 32.77. (9) C24H27O6N1; requires M, 425. Found EIMS m/e
425.1 (M+); Calcd. for C24H27O6N1: C, 67.76; H, 6.35; N, 3.29.
Found: C, 67.77; H, 6.33; N, 3.28. (10) C28H29O6N1; requires M,
475. Found EIMS m/e 475.2 (M+); Calcd. for C28H29O6N1: C,
70.73; H, 6.10; N, 2.94. Found: C, 70.87; H, 5.92; N, 2.93. (11)
C24H29O7N1; requires M, 443. Found EIMS m/e 443.1 (M+); Calcd.
for C24H29O7N1: C, 65.01; H, 6.54; N, 3.16. Found: C, 65.02; H,
6.53; N, 3.11. (12) C28H28O6N1Cl1; requires M, 509. Found EIMS
m/e 509.1 (M+); Calcd. for C28H28O6N1Cl1: C, 66.01; H, 5.05; N,
2.75. Found: C, 71.05; H, 6.12; N, 2.95. (13) C27H28O6N2; requires
M, 476. found EIMS m/e 476.1 (M+); Calcd. for C27H28O6N2: C,
68.06; H, 5.88; N, 5.88; Found: C, 68.09; H, 5.86; N, 5.89. (14)

D. Bartusik et al. / Bioorganic Chemistry 37 (2009) 193–201
201
C26H29O7N2F3; Calcd. for C26H29O7N2F3: C, 57.99; H, 5.39; N, 5.20;
F, 10.59. Found: C, 57.58; H, 5.32; N, 5.28; F, 10.59. (31)
C26H27O7N2F3; Anal. Calcd. for C26H27O7N2F3: C, 58.20; H, 5.03; N,
5.22; F%, 10.36. Found: C, 57.99; H, 5.88; N, 5.28; F, 10.55. (32)
C30H29O7N2F3; Calcd. for C30H29O7N2F3: C, 61.43; H, 4.94; N, 4.77;
F, 9.72. Found: C, 61.71; H, 4.65; N, 4.37; F, 9.49. (33)
C26H29O7N2F3; Calcd. for C26H29O7N2F3: C, 57.99; H, 5.39; N, 5.20;
F, 10.59. Found: C, 56.38; H, 5.21; N, 4.68; F, 9.55. (34)
C30H28O7N2Cl1F3; Calcd. for C30H28O7N2Cl1F3: C, 58.06; H, 4.51; N,
4.50; F, 9.19. Found: C, 58.04; H, 4.29; N, 4.12; F, 8.43. (35)
C26H28O7N2F3; Calcd. for C26H28O7N2F3: C, 58.06; H, 4.86; N, 4.69;
F, 9.56. Found: C, 58.12; H, 4.87; N, 4.69; F, 9.57. (36)
C30H28O7N2Cl1F3; Calcd. for C30H28O7N2Cl1F3: C, 58.06; H, 4.15; N,
4.12; F, 8.41. Found: C, 58.06; H, 4.14; N, 4.13; F, 8.40. (37)
C30H28O7N2Cl1F3; Calcd. for C30H28O7N2Cl1F3: C, 58.06; H, 4.15; N,
4.12; F, 8.41. Found: C, 58.07; H, 4.18; N, 4.12; F, 9.26. (38)
C30H35O7N2F3; Calcd. for C30H35O7N2F3: C, 60.81; H, 5.91; N, 4.72;
F, 9.62. Found: C, 60.79; H, 5.67; N, 4.63; F, 9.67.
are made to discover more effective analogues of colchicine by
modifying basic structure (1).Various arguments e.g. duration of
the exposure to colchicine analogues (2–38), interaction among
cells, drug metabolization may be put forward to explain the dif-
ference in cells’ viability that corresponds to growth inhibition
using prepared analogues. As expected, the fluorinated derivative
(28–38) displayed the high antagonistic potency on cells growth
in 3-D cultures. The use of 1H provides potential tool for the study
of treatment efficacy of the CEM cells. In the studied CEM cells, the
19F SI increased due to 19F uptake, however the cells that are suc-
cessfully treated are no longer viable for trypan blue assays. There-
fore, combined measurements of viability using Trypan blue and
drug uptake using 19F SI gave total cell number that is equal to
the number of cells before treatment.
Considering the applied technique, HPLC has proven particu-
larly effective in the determinations of apoptotic protein even in
low concentrations. Moreover, reversed-phase (RP) HPLC is known
as reliable method for the separation of a great number of proteins
and peptides with high reproducibility. Therefore, we established a
fractionation procedure to enrich less abundant proteins using RP
HPLC. The cells’ viability caused by apoptosis has been suggested
to be a major factor in cell death in treatment of malignancies, such
as lymphoma. In particular, HPLC profile explains why this nonvi-
able cell that expresses specific receptors occurred mostly in trea-
ted cells. It has been also reported that determined Tn antigen is
expressed in over 70% of human carcinoma cells [14].
4. Discussion
Success of drug efficacy to treat cancerous tissues is highly
dependent on their interaction with cells. Currently it is evident
that the efficient drug-cell interaction, accurate drug delivery, traf-
ficking of the therapeutic and the high density cell culture should
be considered for ex vivo studies.
In human body CEM tumor exists in 3-D environment, however,
conventional monolayer cell cultures used in biological and toxico-
logical studies are two dimensional (2-D). Recent pioneering re-
search in the field of cancer has illustrated that 3-D culture
systems can maintain in vivo condition, what is unavailable for
2-D cells suspension [13]. To study the treatment ex vivo, we em-
ployed 3-D structure of cells developed in HFB. The use of HFB al-
lowed to obtain high density cancerous tissue, suitable for MRI
monitoring of the dynamic of the cellular changes.
Colchicine derivatives with substitutions at C-1 position of A
ring are limited in numbers [14]. Thus A ring of colchicine is crucial
for tubulin binding and has attracted attention for further study on
tubulin-microtubule system. For tubulin binding, the size of
methyl group of methoxy substituent of the A ring plays an impor-
tant role. Substitution of methyl group from any of the three-meth-
oxy groups with bulky groups results in manifold reduction of the
potency of cancer cells to grow. As yet, use of colchicine in vivo has
proven to be difficult, as the action has not been completely estab-
lished. We showed that the effect of derivatives presented
improvement of the IC50 values and caused tumor suppression.
The lack of clinical interest in the colchicine (1) arises from its tox-
icity. However, cancer cells are significantly more vulnerable to
colchicine poisoning than healthy cells. Therefore, many attempts
Finally, the study shows, that 19F MRI, HPLC-UV and Trypan
blue assays are suitable for monitoring of cells’ condition before
and after treatments with colchicine derivatives.
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