Synthesis and biological evaluation of colchicine B-ring analogues tethered with halogenated benzyl moieties

Article abstract of DOI:10.1021/jm301151t

Deacetylcolchicine was reacted with substituted benzyl halides to provide a library of compounds for biological analysis. Compound 7 (3,4-difluorobenzyl-N- aminocolchicine) was shown to possess cytotoxicity in cancer cell lines in the low nanomolar range.

Full text of DOI:10.1021/jm301151t

Brief Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Colchicine B‑Ring Analogues
Tethered with Halogenated Benzyl Moieties
Laura Cosentino,†,⊥ Mariano Redondo-Horcajo,∥ Ying Zhao,§ Ana Rita Santos,† Kaniz F. Chowdury,†
Victoria Vinader,† Qasem M. A. Abdallah,†,# Hamdy Abdel-Rahman,†,∞ Jeremie Fournier-Dit-Chabert,†
́
́
Steven D. Shnyder,† Paul M. Loadman,† Wei-shuo Fang,§ Jose Fernando Díaz,∥ Isabel Barasoain,∥
́
Philip A. Burns,‡ and Klaus Pors*,†
†Institute of Cancer Therapeutics, University of Bradford, Bradford, West Yorkshire, BD7 1DP, U.K.
‡Section of Pathology and Tumor Biology, Leeds Institute of Molecular Medicine, St. James’s University Hospital, Leeds, LS9 7TF,
U.K.
§Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1 Xian Nong Tan Street,
Beijing 100050, P. R. China
∥Centro de Investigaciones Biolog
́
icas, CSIC, 28040, Madrid, Spain
S
* Supporting Information
ABSTRACT: Deacetylcolchicine was reacted with substituted benzyl halides to provide a library of compounds for biological
analysis. Compound 7 (3,4-difluorobenzyl-N-aminocolchicine) was shown to possess cytotoxicity in cancer cell lines in the low
nanomolar range. Significantly, it showed no loss of activity in the resistant A2780AD ovarian carcinoma cell line known to
overexpress the ABCB1 drug transporter and was also unaffected by overexpression of class III β-tubulin in HeLa transfected
cells.
the colchicine binding domain will lead to improved therapeutic
index by increasing selectivity for the cellular target, reducing
availability for drug efflux via P-gp and leading to an overall
reduction in toxicity. As an extension of our ongoing efforts to
develop novel analogues that evade traditional resistance
mechanisms and possess improved target selectivity, we here
report on a library of compounds derived from colchicine, which
have been modified to improve the affinity at the colchicine
binding domain.
INTRODUCTION
■
Although microtubule disrupting agents are among the most
successful drugs used in chemotherapy, they are often ineffective
against cancer cells displaying multidrug-resistance (MDR).1
Central to MDR is the membrane-bound P-glycoprotein (P-gp,
ABCB1, MDR1), which is overexpressed in several different
tumor types and known to be associated with poor response to
chemotherapy.2 Resistance to different classes of microtubule
disrupting agents may also be tumor specific. For example, the
Vinca alkaloids are mostly effective against hematological cancers
but often ineffective against solid tumors and taxoids such as
paclitaxel and are effective in treating ovarian, mammary, and
lung tumors but not other solid tumors such as colon
carcinomas.3 Colchicine, the main alkaloid of the poisonous
plant meadow saffron (Colchicum autumnale L.), is used for the
treatment of gout and familial Mediterranean fever.4 Although
colchicine is not clinically used to treat cancer because of toxicity,
it exerts antiproliferative effects through the inhibition of
microtubule formation, leading to mitotic arrest, antivascular
disruption, and cell death by apoptosis.5 It is well-established that
synthetic modifications to the A-ring are associated with loss of
biological activity while there is some tolerance in regard to
manipulation of the C-ring.6 In contrast, modification of the B-
ring at the terminal acetamide has led to interesting accounts
detailing structure−activity relationships (SARs), some aimed at
overcoming MDR in cancer,7−9 while others including ourselves
have investigated ways of delivering high doses to tumor tissue
while not affecting normal tissue.10 Significantly, we have shown
that agents with capacity to covalently bind microtubules possess
properties that are essential in overcoming MDR.11,12 Similarly,
it can be hypothesized that compounds with increased affinity for
RESULTS AND DISCUSSION
■
Chemistry. The synthetic route to the novel compounds
encompassed traditional synthetic modifications. The C7
acetamide group at the B-ring was removed in a three-step
procedure as previously reported.13 Deacetylcolchicine was
reacted with substituted benzyl bromides to afford target
compounds with 40−71% yields (Scheme 1).
Computational Chemistry. The X-ray crystal structure of
bovine α,β-tubulin complexed with N-deacetyl-N-(2-
mercaptoacetyl)colchicine was obtained from the protein
database (PDB code 1SA0, 3.58 Å) as previously described.14
MOE software was employed to generate binding models for 1−
11, which were energy-minimized prior to calculating their
binding affinities. The incorporation of the benzyl fragments at
the deacetylcolchicine B-ring position led to at least 1 order of
magnitude higher affinity in all compounds for the colchicine
tubulin binding site (Table 1). All the colchicine models revealed
Received: August 6, 2012
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
Brief Article
Scheme 1. Synthetic Route to Colchicine B-Modified
Analogues
how the benzyl fragment occupied the chemical space available in
the binding pocket more efficiently than the acetamide group of
colchicine (Figures S1−S11). A previous study focused on
isocolchicine derivatives demonstrated that these were able to
interact with α-tubulin through B-ring substitution in the
colchicine scaffold, thereby increasing their overall binding
affinity for the protein.15 As an example of the binding of the
benzyl tethered colchicines in this study, Figure 1 shows 7
docked into the colchicine binding site, revealing the 3′,4′-
difluorobenzyl moiety to be well accommodated.
Inhibition of Tubulin Assembly. The colchicinoids were
incubated with purified bovine brain tubulin and investigated for
the ability to inhibit tubulin polymerization using conditions
previously described.16 From the data in Table 1, the new
analogues exhibited strong inhibition of tubulin assembly with
5−7 approximately 5 times more potent than colchicine.
Chemosensitivity. The modified colchicines were evaluated
for their ability to inhibit the growth of DLD-1 colorectal cancer
cells. All compounds, bar 10 which precipitated out, displayed
potent cytotoxicity in the low 1−50 nM range. Although data
from computational modeling and the microtubule polymer-
ization assay demonstrated all new compounds to be more affinic
for the colchicine binding site than colchicine, only 6 (3-fold)
and 7 (10-fold) were actually more cytotoxic with 4 and 11 being
equicytotoxic. To obtain further SAR information, we evaluated
the fluorinated compounds within this series (5−9) and the 4′-
OCH3 (11) analogue in the HT29 colon cancer cell line, and the
more aggressive MDA-MB-231 breast cancer cell line. Generally,
the compounds were found to possess similar potency in the
HT29 cells as was observed for the DLD-1 cells, but all
Figure 1. Compound 7 docked into the colchicine binding site.
compounds including colchicine lost 3- to 20-fold potency in the
MDA-MB-231 cells. Within the fluorine series it was apparent
that the monosubstituted 5 or compounds containing fluorine in
the 5′-position of the attached benzyl moiety (8 and 9) lost
potency compared to 2′,3′- (6) or 3′,4′- (7) disubstituted
analogues, which is noteworthy given that the computational
modeling did not predict these empirical data. Compound 7 was
shown to be consistently more potent than colchicine, exhibiting
low nanomolar cytotoxicity (1−15 nM) across the three cancer
cell lines investigated; hence, it was selected for further studies.
Cell Cycle and Microtubule Disruption. Exposure of
HT29 cells to 7 led to accumulation of cells in the late G2/M
phase after 24 h (92.5%) or 48 h (91.2%), which was not
observed for untreated control samples (12.7% and 8.8%,
respectively), revealing similar cell cycle profile to colchicine but
with a more potent agent (Figure S12). To evaluate the effect on
endothelial cell morphology and cytoskeletal organization, HT29
cells were exposed to 7 for 24 h followed by tubulin
immunostaining. Analysis of the results revealed changes to the
cell shape and disruption of the microtubule network in a dose-
dependent manner (Figure 2).
Drug Resistance. Although 7 is more potent than colchicine
across the three cancer cell lines, the mechanism for this
biological activity appeared to be similar, given the experimental
observations from the cell cycle and morphological studies.
Indeed, the increased activity could be associated with higher
Table 1. Physicochemical Properties, Tubulin Binding Affinities, and Growth Inhibition against a Human Cancer Cell Lines
a
b
c
d
compd
R
MW
CLogP
affinity [pKi]
ITA IC50 [μM]
DLD-1 IC50 [nM]
HT-29 IC50 [nM]
69.25 6.71
MDA-MB-231 IC50 [nM]
37 1.41
colchicine
H
399.4
447.5
526.4
465.2
482.0
573.4
483.5
483.5
483.5
501.5
492.5
477.6
2.586
4.506
5.269
4.645
5.160
5.111
4.784
4.784
4.784
4.923
4.414
4.515
8.792
9.435
9.897
9.553
9.737
9.914
9.703
9.846
9.882
10.196
10.233
10.337
7.85 0.89
15.6 0.46
31.17 1.26
32.60 0.66
35.90 1.10
11.38 0.41
51.6 5.33
4.67 0.15
1.61 0.13
35.2 0.80
36.2 0.25
e
e
e
1
4′-Br
4′-Cl
ND
ND
ND
e
e
2
1.70 0.20
ND
ND
e
e
e
3
4′-I
ND
ND
ND
e
e
e
4
4′-F
3′-F
ND
ND
ND
5
1.49 0.18
1.62 0.15
1.52 0.18
2.98 0.29
4.60 0.42
1.99 0.21
6.49 0.68
63.4 2.3
7.43 5.73
4.3 1.14
52 1.2
89.1 1.3
86 3.47
16 2.22
523 5.34
92.2 1.95
6
2′,3′-F
3′,4′-F
3′,5′-F
3′,4′,5′-F
4′-NO2
4′-OCH3
7
8
9
64.5 1.03
f
e
e
10
11
Prec
ND
ND
15.7 0.59
51 3.13
93 6.06
a
b
c
CLogP was calculated using the Wildman and Crippen model. The calculated binding affinity was generated using MOE software. Inhibition of
d
tubulin assembly (IC50 = concentration required to inhibit 50% of the DAPI-fluorescence enhancement due to tubulin polymerization). IC50 values
are the mean SD of at least three independent assays. ND = not determined. Prec = compound precipitation, no IC50.
e
f
B
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Journal of Medicinal Chemistry
Brief Article
Figure 3. Effects of 7 and colchicine on the induction of ABCB1 and
ABCG2 transporter genes in DLD-1 (A) and HT29 (B) cells.
the A270AD cells and it was not judged a good substrate for
ABCG2 in A549 cells (known to express the highest levels of
ABCG2 function among cells in the NCI 60 human cancer cell
line screen19) given its cytotoxicity was not affected by the
presence of the ABCG2 inhibitor fumitremorgin C (Figure S14).
Tubulin Resistance. A recent study20 disclosed evidence
that several agents able to bind at the colchicine site of tubulin
were unaffected by class III β-tubulin resistance, indicating that
new compounds affinic for this site could play a role as an
alternative option to taxoids against patients that relapse because
of drug-resistant tumors. In this respect, we also evaluated 7 in
parental HeLa cells and a transfected β-III resistant variant.21 The
latter is resistant to agents binding at the paclitaxel binding site
and the Vinca alkaloid binding site.22 As the data in Table 3
Figure 2. Disruption of the microtubules. HT29 cells were exposed to 7
at 5 (A), 50 (B), and 500 nM (C) for 24 h and compared to untreated
cells (D). Microtubules are stained in red. Chromatin is in blue.
lipophilicity and more efficient cellular uptake of 7 (log P = 4.78)
compared with colchicine (log P = 2.586). Colchicine undergoes
intensive hepatic metabolism with B-ring deacetylation through
CYP3A4, and the excretion is largely dependent on P-gp.17
Incubation of 7 with mouse liver homogenate revealed no
breakdown products after 2 h, demonstrating that modification
of the acetamide with a 3,4-difluorobenzyl moiety provides
stability to the compound (Figure S13). Evaluation of 7 in the
A2780 ovarian carcinoma cell line and the P-gp overexpressing
A2780AD variant showed that this compound was unaffected by
P-gp expression. In contrast, colchicine and paclitaxel lost 2.5-
and 900-fold in potency, respectively (Table 2).
Table 3. Growth Inhibition of HeLa Parental and HeLa β-III
Transfected Cervix Carcinoma Cells by 7, Colchicine, and
Paclitaxel
a
IC50 [nM]
b
compd
HeLa
HeLa βIII
RF
Table 2. Growth Inhibition of A2780 Parental and A2780AD
Resistant Ovarian Carcinoma Cells by 7, Colchicine, and
Paclitaxel
7
25.3 0.8
8.2 0.3
1.0 0.1
14.1 0.8
9.7 0.3
11.2 0.1
0.55
1.2
colchicine
paclitaxel
11.2
a
a
IC50 values are the mean
standard error of at least four
IC50 [nM]
b
independent assays. The resistance factor (RF) = IC50 of the
resistant HeLa β-III cell line/IC50 of the HeLa cell line.
b
compd
A2780
A2780AD
RF
7
18.8 1.4
6.7 0.4
17.2 1.4
16.8 1.1
>1000
0.9
colchicine
paclitaxel
2.5
b
reveal, 7 not only is equipotent in the β-III tubulin transfected
cancer cell line but has an almost 2-fold increased activity. The
reason for this might be that transfected HeLa cells overexpress
class III β-tubulin, which may induce an increase in microtubule
dynamics. This would explain why agents binding at the
colchicine site are working more efficiently compared with
microtubule stabilization agents such as the taxoids.
1.1 0.5
>900
a
IC50 values are the mean
standard error of at least four
independent assays. The resistance factor (RF) = IC50 of the
A2780AD cell line/IC50 of the parental A2780 cell line.
b
Induction of ABC Transporter Proteins. In addition to
using the ABCB1-overexpressing A2780AD cell line, further
studies evaluated whether colchicine or the new analogue 7
would induce this transporter or ABCG2 (BCRP1). The latter is
another transporter protein that has been shown to cause
resistance to drugs such as camptothecin and mitoxantrone.2
Given the lack of clinical success of taxoids in the treatment of
colon carcinomas, HT29 and DLD-1 cell lines were utilized. To
determine the effect on gene transcript level, cells were exposed
for 96 h to concentrations equal to IC50 or 1/10 of the IC50. QRT-
PCR was used to quantitatively examine gene expression levels
by combining with reverse transcription and measuring mRNA
levels for the two ABC genes. The results shown in Figure 3
revealed that induction at the gene level is possible in the HT29
cells but not in the DLD-1 cells, suggesting that long-term
exposure may lead to resistance via ABCB1 and ABCG2 in the
former cell line. In contrast, the short-term chemosensitivity
assays revealed that 7 was not affected by high levels of ABCB1 in
CONCLUSION
■
Despite of the emergence of molecularly targeted agents,
microtubule-targeting agents will continue to offer physicians
viable therapeutic options to combat cancer in the foreseeable
future.3,23 However, no compounds based on the colchicine
scaffold have been successful in treating cancer patients. Many
factors related to the colchicine architecture warrant further
exploration including the structural simplicity of the scaffold, the
availability of the natural product, the ease with which this natural
product can be synthetically manipulated, the ability to down-
regulate several drug-metabolizing cytochrome P450 enzymes18
and hence avoid phase I metabolism, and ability to evade class III
β-tubulin resistance. Our ongoing synthetic and biological
studies on several different classes of anticancer agents are
aimed at combining properties that make cytotoxics and
prodrugs evade conventional resistance mechanisms but also
C
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Journal of Medicinal Chemistry
Brief Article
(all from Sigma). A2780 and A2780AD ovary carcinoma cell lines were
supplemented as well with 0.25 units/mL bovine insulin. HeLa and
HeLa β-III21 cervix carcinoma lines were grown in DMEM
supplemented with sodium pyruvate, glutamine, and fetal bovine
serum as above, but the last was additionally supplemented with 0.5 mg/
mL G418 sulfate. An amount of 1 ×104 cells/mL was inoculated into
each well of a 96-well plate and incubated overnight at 37 °C in a
humidified atmosphere containing 5% CO2. Compounds were dissolved
in DMSO and diluted in complete cell culture medium to give a broad
range of concentrations such that the final DMSO concentration was
not greater than 0.1%. Medium was removed from each well and
replaced with compound or control solutions. After 96 h of incubation,
the MTT assay was performed to assess chemosensitivity.
Immunocytochemical Analysis of Microtubule Disruption.
HT29 cells at 70% confluency were trypsinized and transferred into six-
well plates (5 × 105 cells/well) and allowed to adhere overnight at 37 °C
in a humidified incubator prior to the treatment, and the
immunocytochemical analysis was performed as previously described.27
In brief, cells were seeded onto sterilized glass coverslips in six-well
plates (Corning Glass Works, NY, U.S.). Cells were left to adhere for 24
h. Compound 7 (at 5, 50, and 500 nM) was added to each culture for 24
h. Medium was removed, and the cells were fixed in precooled methanol
at −20 °C for 30 min. After washes in PBS, cells were incubated in the
primary monoclonal antibody, mouse anti-α-tubulin (Sigma) at a
dilution of 1:500 in PBS for 30 min at rt. After washes in PBS, the
secondary antibody, TRITC-conjugated rabbit anti-mouse IgG (Dako,
Ely, U.K.) was added at a dilution of 1:50 for 30 min. After final washes,
the cultures were mounted in Vectashield (Vector Laboratories,
Peterborough, U.K.) and stored at 4 °C until analysis. Cells were
analyzed and images captured with a Zeiss LSM510 confocal system
attached to an Axiovert 200 M inverted microscope using LSM510
software (all from Zeiss, Welwyn Garden City, U.K.).
Induction of ABC Transporter Genes. HT29 and DLD1 cell lines
were seeded at 5 × 105 cells/dish and incubated overnight. Cells were
treated for 96 h with selected concentrations of colchicine and 7,
respectively. Total RNA was isolated from the cells using RNeasy Mini
Kit (QIAGEN) according to the manufacturer’s protocol. The RNA
concentration and purity were determined using a microvolume
spectrophotometer. Reverse transcription was performed using multi-
scribe reverse transcriptase kit (Applied Biosystems). The ABCB1 and
ABCG2 mRNA expression levels were quantified using the Stratagene
Mx3005P QPCR system (Agilent Technologies). Quantitative RT-PCR
was carried out using TaqMan Universal PCR master mix (Roche
Applied Biosystems) and specific primer sets: ABCB1 (5′-
CCATAGCTCGTGCCCTTG-3′/5′-AGGGCTTCTTGGACA-
ACCTT-3′) and ABCG2 (5′-TTCCACGATATGGATTTACGG-3′/
5′-GTTTCCTGTTGCATTGAGTCC-3′). Normalization was per-
formed using PPIA as a human endogenous control (FAM/MGB
Probe, Non-Primer Limited, Roche Applied Biosystems). Three
independent PCR analyses were performed. Fold induction was
expressed as the relative ratio (signal of target)/(signal of PPIA) to
the ratio of that in the control in an individual experiment. The
significance of results was assessed through a comparison of mean values
using a paired two-tailed t test with two-way ANOVA followed by
Bonferroni correction for multiple testing. Statistical analysis was
performed using GraphPad Prism version 4.00 (www.graphpad.com).
exploiting the tumor microenvironment to deliver sufficient
doses of compound to elicit an efficient therapeutic
response.10,24,25 We have reported here a new library of
colchicine analogues modified at the B-ring by incorporation of
aryl-substituted fragments, typified by 7, which in comparison
with colchicine possesses improved biological activity against
sensitive and resistant cancer cells in vitro. This study is
important in identifying novel leads for further development of
agents that can evade conventional resistance mechanisms,
thereby improving on the therapeutic index of colchicine.
EXPERIMENTAL SECTION
■
All chemicals were obtained from Aldrich and Lancaster. Solvents were
supplied by VWR bar anh THF (Aldrich). For column chromatography,
silica particle size was 35−70 μm. Thin layer chromatography plates (on
aluminum) were supplied by VWR. H and 13C NMR spectra were
1
measured on a Bruker Advance AM 400 (400 MHz) spectrometer, and
low resolution mass spectra were generated using a Micromass Quattro
Ultima mass spectrometer. The purity of all compounds was ≥95% as
measured by HPLC analysis. A representative example of target
compound synthesis is described below.
3,4-Difluorobenzyl-N-aminocolchicine (7). To a stirring sol-
ution of N-deacetylcolchicine (50 mg, 0.140 mmol) in anhydrous THF
(3 mL) under argon atmosphere were added 3,4-difluorobenzyl
bromide (43.5 mg, 0.210 mmol) and NEt3 (39 μL, 0.280 mmol). The
resulting solution was heated at 65 °C for 16 h. The reaction mixture was
then poured in saturated NaHCO3 (20 mL) and extracted with CH2Cl2
(3 × 25 mL). The combined organic extracts were dried over MgSO4,
and the solvent was removed under vacuum. The crude oil was subjected
to column chromatography on silica gel (petroleum ether/EtOAc 1:1 →
EtOAc) to give the title compound as a yellowish solid (44 mg, 65%). 1H
(400 MHz, CDCl3): 1.61−1.74 (m, 1H, H6), 2.17−2.26 (m, 1H, H6′),
2.34−2.42 (m, 1H, H5), 2.47 (dd, 1H, J 6.1 and 13.6 Hz, H5′), 3.37−
3.42 (m, 2H, H7 and H14), 3.57 (s, 3H, OCH3), 3.68 (d, 1H, J 13.5 Hz,
H14′), 3.90 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.00 (s, 3H, OCH3),
6.52 (s, 1H, HAr), 6.81 (d, 1 H, J 10.7 Hz, HAr), 6.93−7.10 (m, 3 H,
HAr), 7.23 (d, 2 H, J 10.7 HAr), 7.80 (s, 1H, HAr). 13C (101 MHz,
CDCl3): 30.2 (C5), 38.7 (C6), 50.6 (C14), 56.0 (OCH3), 56.3 (OCH3),
59.9 (C7), 60.8 (OCH3), 61.2 (OCH3), 107.1 (CHAr), 111.9 (CHAr),
116.9 (d, J 9.4 Hz, CHAr), 117.0 (d, J 9.4 Hz, CHAr), 123.9 (dd, J 3.5
and 6.0 Hz, CHAr), 125.3 (CAr), 132.0 (CHAr), 134.9 (CHAr), 135.0
(CAr), 136.6 (brs CAr), 136.9 (CAr), 141.1 (CAr), 148.1 (d, J 12.6 Hz,
CAr), 148.9 (d, J 12.7 Hz, CAr), 150.5 (d, J 12.3 Hz, CAr), 150.6 (Car,)
151.3 (d, J 12.5 Hz, CAr), 153.3 (CAr), 163.9 (CAr), 179.7 (CO). m/z
(ES+) 484 [M + H]+ (100%). HMRS calcd for C27H28O5N1F2
484.1930, found 484.1934 [M + H]+.
Molecular Modeling. By use of the information from the X-ray
structure (PDB code 1SA0), mercaptocolchicine was modified to
colchicine and 1−11, following minimization, binding energy estimates
were calculated using a previously described method.14
Inhibition of Tubulin Assembly. Tubulin containing microtubule-
associated protein (MAP) was isolated from pig brain by assembly/
disassembly cycles.26 Compounds were evaluated for inhibition of the
polymerization of MAP-tubulin with minor modification to previous
protocol.16 Briefly, reaction mixtures containing PMEG buffer (0.1 M
PIPES, 1 mM MgSO4, 2 mM EGTA, 10% glycerol, 0.1 mM GTP, pH
6.9), 10 μM MAP-tubulin, and 10 μM DAPI were prepared at 0 °C and
200 μL was added per well in a 96-well plate. Compounds were added at
appropriate concentrations (0.2−50 μM). Fluorescence data were
collected using TECAN Infinite F200 plate reader (excitation 360/35,
emission 465/35) at 37 °C for 60 at 1 min intervals. The IC50 was
obtained by the software GraphPad Prism 4.00 using the following
ASSOCIATED CONTENT
■
S
* Supporting Information
Synthesis details and images of binding models for 1−11; cell
cycle, liver homogenate stability, and chemosensitivity in A459 in
the absence/presence of fumitremorgin C for 7. This material is
available free of charge via the Internet at http://pubs.acs.org.
nonlinear regression fitting equation: ΔF = ΔFmax[drug]/(IC50
+
[drug]). At least three independent determinations were performed for
each compound.
AUTHOR INFORMATION
■
Corresponding Author
Chemosensitivity. The cell lines DLD-1, HT29, and MBD-DA-431
were cultured in RPMI 1640 cell culture medium supplemented with 1
mM sodium pyruvate, 2 mM L-glutamine, and 10% fetal bovine serum
*Phone: +44 (0)1274 236482. Fax: +44 (0)1274 233234. E-
mail: k.pors1@bradford.ac.uk.
D
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Journal of Medicinal Chemistry
Brief Article
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Present Addresses
⊥Fox Cancer Center, 333 Cottman Avenue, 19111, Philadelphia,
PA, U.S.
#Faculty of Pharmacy, Taif University, Taif, Al-Haweiah, P.O.
Box 888, Taif 21974, Saudi Arabia.
∞Department of Medicinal Chemistry, Faculty of Pharmacy,
Assiut University, 71515 Assiut, Arab Republic of Egypt.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
K.P. was supported by YCR Program Grant, EPSRC Science
Bridge (Grant EP/G042365/1), FCT (Grant SFRH/BD/
46871/2008) and J.F.D. by Grant BIO2010-16351 from
Ministerio de Economia y Competitividad and Grant S2010/
BMD-2457 BIPEDD2 from Comunidad Auton
Spain. Parental HeLa and transfected β-III cervix carcinoma cell
lines were a kindly gift from Prof. Richard F. Luduena
(Biochemical Dept., The University of Texas Health Science
Center, TX, U.S.). We thank the EPSRC NMSSC for HRMS
data.
́
oma de Madrid,
̃
ABBREVIATIONS USED
DAPI, 4′,6-diamidino-2-phenylindole; DMEM, Dulbecco’s
modified Eagle medium; MAP, microtubule-associated protein
■
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