Synthesis of combretastatin A-4 O-alkyl derivatives and evaluation of their cytotoxic, antiangiogenic and antitelomerase activity Dedicated to Professor G. Asensio, University of Valencia, on the occasion of his 65th birthday

Article abstract of DOI:10.1016/j.bmc.2013.09.064

We here report the synthesis and biological evaluation of several combretastatin A-4 derivatives alkylated at the phenol hydroxyl group. Some of these derivatives contain an (E)-arylalkene fragment reminiscent of that present in some natural stilbenes like resveratrol. The cytotoxicities towards one human healthy kidney embryonic and two tumoral cell lines were determined. In addition, the ability of these compounds to inhibit the production of the vascular endothelial growth factor (VEGF) was measured. Finally, the expression of genes controlling the production of telomerase was measured. Some of the compounds were found to have an activity comparable or higher than that of combretastatin A-4 in at least one of the aforementioned biological properties. The compounds with the (E)-arylalkene fragment were in general terms more active than the simple O-alkyl derivatives. However, no clear structure/activity correlations were perceived when comparing the observed compound activities across the three biological properties. This points out the existence of marked differences between the mechanisms responsible for their cytotoxicity.

Full text of DOI:10.1016/j.bmc.2013.09.064

Bioorganic & Medicinal Chemistry xxx (2013) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of combretastatin A-4 O-alkyl derivatives and evaluation
of their cytotoxic, antiangiogenic and antitelomerase activity
a
a
Sandra Torijano-Gutiérrez a, Santiago Díaz-Oltra a, Eva Falomir a, , Juan Murga , Miguel Carda ,
⇑
J. Alberto Marco b,
⇑
a Depart. de Q. Inorgánica y Orgánica, Univ. Jaume I, E-12071 Castellón, Spain
b Depart. de Q. Orgánica, Univ. de Valencia, E-46100 Burjassot, Valencia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history:
We here report the synthesis and biological evaluation of several combretastatin A-4 derivatives
alkylated at the phenol hydroxyl group. Some of these derivatives contain an (E)-arylalkene fragment
reminiscent of that present in some natural stilbenes like resveratrol. The cytotoxicities towards one
human healthy kidney embryonic and two tumoral cell lines were determined. In addition, the ability
of these compounds to inhibit the production of the vascular endothelial growth factor (VEGF) was
measured. Finally, the expression of genes controlling the production of telomerase was measured. Some
of the compounds were found to have an activity comparable or higher than that of combretastatin A-4 in
at least one of the aforementioned biological properties. The compounds with the (E)-arylalkene
fragment were in general terms more active than the simple O-alkyl derivatives. However, no clear struc-
ture/activity correlations were perceived when comparing the observed compound activities across the
three biological properties. This points out the existence of marked differences between the mechanisms
responsible for their cytotoxicity.
Received 19 June 2013
Revised 22 September 2013
Accepted 26 September 2013
Available online xxxx
Dedicated to Professor G. Asensio,
University of Valencia, on the occasion of his
65th birthday
Keywords:
Anticancer drugs
Cytotoxic compounds
Antiangiogenic compounds
VEGF
Ó 2013 Elsevier Ltd. All rights reserved.
Telomerase
Combretastatin A-4 derivatives
1. Introduction
of VEGF has been reported to occur in various types of tumors.9
Not unexpectedly, VEGF has become one further key target mole-
It is widely known that cancer, one leading cause of death in
developed countries,1 may be induced by a plethora of both exter-
nal and internal factors, including genetic mutations. Accordingly,
a number of types of therapeutic attack has been investigated.2,3
One of these involves the use of cytotoxic drugs, which exert their
effect in many cases by means of inducing cell apoptosis.4 A second
type of therapeutic strategy against cancer is based on the use of
compounds with vascular-targeting properties. These may be due
to their ability to either inhibit the formation of new blood vessels
(antiangiogenic agents) or else to promote the destruction of exist-
ing ones (antivascular agents).5,6 Tumor angiogenesis is a very
complex process and involves the tight interplay of many factors.7
One of these is a protein called vascular endothelial growth factor
(VEGF), a key regulator of angiogenesis which promotes endothe-
lial cell survival, proliferation and migration while increasing
vascular permeability.8 In fact, overexpression in the production
cule in cancer therapy.10,11
A third therapeutic line follows the path of the chromosomal
telomeres.12 Telomeres are the terminal zones of chromosomes
and display a special structure that fulfills at least two essential
functions: (a) they must be recognized as functional domains, thus
distinguishing them from random chromosomal breaks that would
stimulate the onset of repair mechanisms; (b) they must prevent
these DNA ends from fusing with other DNA ends.13 To comply
with these functions, an appropriate amount of repetitive DNA
sequences (telomeres) must be added to the ends of the chromo-
somes. This task is fulfilled by a special type of ribonucleoprotein
complex called telomerase, an enzyme with reverse transcriptase
activity. The expression of this enzyme is restricted or absent in
normal human somatic cells and so telomeres progressively short-
en during cell lifespan.12 This triggers a DNA damage response
which culminates in cell senescence or apoptosis.14
Shortening of telomeres during cell division provides a barrier
for tumor progression. Indeed, cancer cells have evolved the ability
to overcome senescence by using mechanisms capable of main-
taining telomere lengths, such as expressing telomerase, which
enables them to divide indefinitely. This uncontrolled cell growth
⇑
Corresponding authors. Fax: +34 964 728214 (E.F.); tel.: +34 96 3544337; fax:
+34 96 3543880 (A.M.).
E-mail addresses: efalomir@qio.uji.es (E. Falomir), alberto.marco@uv.es
(J.A. Marco).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.064
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2
S. Torijano-Gutiérrez et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
is a key feature of cancer malignancy and has been found to corre-
late with telomerase reactivation.15 For this reason, and since telo-
merase has been detected in about 90% of all malignant tumors,12
drugs with ability to inhibit telomerase activity are potentially use-
ful weapons in the fight against cancer, aging and other diseases,
including some related to premature telomere shortening.16
The emergence of resistances to existing drugs has led to a
continuous need of developing new bioactive compounds that
overcome such problems. Even though first observed in the case
of antibiotics, resistances have been reported to therapies with
various types of cytotoxic,17 antiangiogenic18 and antitelomerase
agents.19 The discovery and investigation of new members of
these compound classes therefore constitutes an important goal
in chemistry and pharmacology. Among the new drug types, the
combretastatin family, which belongs like the well-known resve-
ratrol to the stilbene class of natural products,20 has acquired an
outstanding status in the last years and their members have been
found utility in various pharmacological applications.21 The
knowledge of the chemistry, biology, and medical potential of this
particular compound class, discovered about 30 years ago in the
African tree Combretum caffrum, has continued to advance.22 Pre-
clinical and clinical developments over the last decade have been
rapidly accelerating for drugs such as combretastatin A-4 (1a),
most particularly in the form of its phosphate prodrug CA4P
(1b), and for combretastatin A-1 (1c) as promising cancer vascu-
lar-disrupting and ophthalmology drugs (Fig. 1).22,23 Indeed, these
encouraging developments have stimulated a variety of efforts
devoted to the synthesis and biological evaluation of numerous
combretastatin structural modifications.24 Recent reports include
SAR studies that provide varying levels of cancer cell growth
inhibition.25
MeO
MeO
2a
2b
R = CH2CH=CH2
R = (E)-CH2CH=CHMe
2c R = CH2Ph
2d R = p-CH2C6H4OMe
OMe
OMe
OR
2e
R = p-CH2C6H4Br
OMe
OMe
2f Ar = o-MeOPh
2g
Ar = m-MeOPh
MeO
MeO
Ar
2h Ar = p-MeOPh
2i Ar = o-MOMOPh
2j
2k
Ar = m-MOMOPh
Ar = p-MOMOPh
O
2l Ar = p-FPh
2m Ar = p-ClPh
2n
Ar = p-BrPh
Figure 2. Structures of combretastatin A-4 O-alkyl derivatives 2a–2n used in this
study (MOM = MeOCH2).
observed results might be possibly helpful in aiding to clear the
mechanisms of action of these compounds.
3. Chemical results
Combretastatin A-4 1a, the starting material for all compounds
discussed here, was prepared according to a literature procedure.27
Conversion into O-alkyl derivatives 2a–2n was performed as
depicted in Scheme 1 using bromides 3a–3n (Scheme 2) as the
alkylating reagents. Yields are given in the experimental part.
From the alkyl bromides mentioned in Scheme 1, 3a–3e are
commercially available. Bromides 3f–3n were synthesized by
means of ruthenium-catalyzed cross metathesis28 between the
commercially available bromide 4 and styrene derivatives 5–13
(Scheme 2). Yields are given in the experimental part.
2. Research purpose
4. Biological results
In the present paper, we are disclosing our results in the synthe-
sis and biological evaluation of a number of O-alkyl derivatives of
combretastatin A-4 1a. These are arranged in two series as shown
in Fig. 2. One includes compounds 2a–e having O-alkyl residues of
the allyl and benzyl type. The second series encompasses com-
pounds 2f–n, which contain (E)-5-arylpent-4-enyl residues with
various aryl groups. This part of the structure displays an arylalk-
ene fragment which shows similarity to a part of the structure of
antiangiogenic stilbenes of the resveratrol type (Fig. 1).20,26
Accordingly, it may be expected that these compounds could not
only exhibit cytotoxicity but also antiangiogenic activity. Thus,
we here present the results of our measurements of the cytotoxic-
ities of compounds 2a–n but also of their ability to inhibit the
production of the VEGF. Finally, and in order to cover all three
aforementioned lines of therapeutic attack, we also have tested
their ability to inhibit telomerase activity. In our belief, the
4.1. Cytotoxicity of combretastatine derivatives
We first carried out a measurement of the cytotoxicity of the
synthetic combretastatin derivatives 2a–2n. To this purpose, MTT
assays were performed using two tumoral cells, the human colon
HT-29 and the breast adenocarcinoma MCF-7 cell lines, as well
as one normal cell line, the human embryonic kidney cell line,
HEK-293.29 Cytotoxicity values, expressed as the compound con-
centration (lmol/L) that causes 50% inhibition of cell growth
(IC50), are shown in Table 1. The observed values are in the low
to medium micromolar range, with compound 2i showing the low-
est values, not very different of those of combretastatin A-4 (CoA4)
for these two particular cell lines. In addition, it is worth mention-
ing that some of the synthetic compounds are much more toxic for
tumoral cells than for normal ones, an obviously desirable feature.
This can be better appreciated with the
a and b coefficients, ob-
tained by dividing the IC50 values of the normal cell line by those
of one or the other tumoral cell line (see footnote in Table). The
highest value of either coefficient, the highest the therapeutic
safety margin of the compound in the corresponding cell line. Thus,
combretastatin A-4 shows high values of both coefficients, most
particularly in the case of the MCF-7 cell line. This turns out also
MeO
1a R1 = R2 = H
R1
1b
1c
R1 = H R2 = PO3Na2
R1 = OH R2 = H
MeO
OMe
OR2
OMe
OH
MeO
HO
RBr (3a-3n), K2CO3
MeO
1a
Resveratrol
2a-2n
OMe
DMF, RT, 24 h
OR
OH
Figure 1. Structures of some combretastatins (1a–1c) and resveratrol.
OMe
Scheme 1. Synthesis of combretastatin A-4 O-alkyl derivatives 2a–2n.
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S. Torijano-Gutiérrez et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
3
3c R = H
Br
3a
3b
R = H
R = Me
R
Br
3d R = OMe
3e R = Br
R
Ru-II
, CH2Cl2
Ar
Ar
+
Br
Δ
, 18 h
Br
3f
5 Ar = o-MeOPh
4
Ar = o-MeOPh
6
7
3g Ar = m-MeOPh
3h Ar = p-MeOPh
Ar = m-MeOPh
Ar = p-MeOPh
3i
8 Ar = o-MOMOPh
9 Ar = m-MOMOPh
Ar = o-MOMOPh
ArN
NAr
Ph
3j Ar = m-MOMOPh
3k Ar = p-MOMOPh
10
11
Ar = p-MOMOPh
Ar = p-FPh
Cl
Cl
Ru
3l
3m
Ar = p-FPh
Ar = p-ClPh
PCy3
Ru-II
12 Ar = p-ClPh
13 Ar = p-BrPh
3n Ar = p-BrPh
(Ar = 2, 6-diisopropyl-
phenyl)
Scheme 2. Synthesis of alkyl bromides 3f–3n.
Figure 3. VEGF protein production from HT-29 cells treated with DMSO (control),
combretastatin A-4 (CoA4) and derivatives 2a–n (at least three measurements were
performed in each case). Bars represent mean values of VEGF expression (in ng/mL)
and error bars indicate standard errors of the mean. The statistical significance was
evaluated using one-sample t-tests (P <0.001).
Table 1
Cytotoxicity of combretastatin A-4 derivatives 2a–2na
Compound
HT-29
MCF-7
0.2
12.0 0.4
5.4 0.4
HEK-293
25
43.5 0.8
135 13
ab
bc
CoA4
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
4.2 0.5
1
3
5.9
1.5
0.8
0.4
1.4
0.4
>4.6
1.6
1.9
11
0.5
3.6
1.2
6.2
5.0
25
3.6
25
47% of the control value. Similarly strong effects were observed
in the cases of compounds 2b (44%) and 2i (42%). The remaining
compounds showed less favourable values, with compound 2e
being essentially inactive (99%).
29
3
161
39
47
5
6
5
59
48
29
21
41
16
2
1
1
2
3
3
15
64
45
3
7
4
0.3
1.3
1.6
>19
1.0
5.5
24.1
3.7
0.6
7.2
2.8
1.5
108
86
25
1
While the results depicted in Figure 3 point out that compounds
2a–n cause inhibition of the VEGF production, they do not say any-
thing about the precise phase of the VEGF generation process with
which they interfere. In order to deepen into the knowledge of this
issue, we proceeded to determine whether the compounds under
study were able to control protein production at the transcriptional
level. With this idea in mind, we performed a reverse transcriptase/
polymerase chain reaction (RT-PCR) analysis. For this purpose, we
selected six of the combretastatin A-4 derivatives with the highest
anti-VEGF activities (2b, 2g, 2i, 2j, 2k and 2n). As shown in Figure 4,
treatment of HT-29 tumoral cells with these derivatives and with
combretastatin A-4 in DMSO did in fact cause a reduction of the
transcription of VEGF mRNA as compared with control cells (the
values of these are standardized to 100%). The most active deriva-
tives turned out to be 2b and 2k, which proved able to reduce the
expression of the corresponding gene to less than 70% of the con-
trol value. These effects are thus stronger than that of combretast-
atin A-4 itself, which only causes a slight reduction of the
transcription level of VEGF mRNA (91%). Interestingly, compound
2g, which showed the highest degree of inhibition of VEGF produc-
tion (Fig. 3), leaved the transcription of VEGF mRNA practically
unaltered (97%).
6
3
7
>400
39
2
47
88
6
9.6 0.2
4.4 0.8
106
30
25
9
2
4
59
7
98
18
11
7
0.4
8
3
2
8
3
42
16
40
37
7
2
5
8
115
111
55
5
3
5
Values of
a and b have been rounded off to a decimal figure. The lowest IC50 values
(for tumoral cells) and the highest values of the
a and/or b coefficients (>4) have
been highlighted in italics.
a
IC50 values include those of combretastatin A-4 itself CoA4 and are expressed as
mol/L) that causes 50% inhibition of cell growth,
and are the average ( SD) of three different measurements (described in Section 6).
the compound concentration (
l
b
a
= IC50 (HEK-293)/IC50 (HT-29).
c
b = IC50 (HEK-293)/IC50 (MCF-7).
to be the case of compounds 2f and 2i. Compounds 2m and 2n
show a good selectivity only in the case of the HT-29 line ( >4)
whereas compounds 2b, 2h and 2l show a good selectivity in the
a
specific case of the MCF-7 line (b >4).
4.2. Effect of combretastatin A-4 derivatives on VEGF
production
Table 2 present the same results depicted in Figures 3 and 4 for
the aforementioned six compounds (2b, 2g, 2i, 2j, 2k and 2n)
although in the form of percentages of inhibition of VEGF produc-
tion (100—% VEGF production) and of gene expression (100—%
gene expression): thus, the highest values correspond to the stron-
gest inhibitory effect. There is a visible lack of parallelism between
the data of protein inhibition and those of gene inhibition. This
suggests that these combretastatin A-4 derivatives exert the
control of VEGF production at a phase different from that of gene
transcription, perhaps during the posttranslational stage.
According to that discussed in the Introduction section, we next
investigated whether combretastatin derivatives 2a–n were able to
inhibit or at least decrease the production of the VEGF protein in
HT-29 tumoral cells. Figure 3 shows the results of VEGF production
obtained by means of ELISA measurements after treatment of
HT-29 tumoral cells with combretastatin A-4 and with compounds
2a–n dissolved in DMSO, which was the control substance in all
experiments (in all cases, concentrations values below IC50 were
used). With the control substance, the observed VEGF production
was standardized to 100%, the other values being then referred
to it. Thus, compound 2g caused the highest degree of inhibition
in the VEGF production, which underwent a reduction to 38% of
the control value. This is an even stronger effect than that caused
by combretastatin A-4, where VEGF production was reduced to
4.3. Effect of combretastatin A-4 derivatives on telomerase
production
We were also interested in designing compounds with the
ability to inhibit the expression of telomerase in tumoral cells.
The role of this important ribonucleoprotein has been referred to
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S. Torijano-Gutiérrez et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
investigated their ability to inhibit the expression of the hTERT
and c-myc genes.
In order to determine whether O-alkyl derivatives of combre-
tastatin A-4 were able to regulate the expression of the hTERT
and c-myc genes, we have performed a RT-PCR analysis using
HT-29 tumoral cells. For that purpose, we selected the same group
of derivatives (2b, 2g, 2i, 2j, 2k and 2n) previously investigated for
their antiangiogenic activity. The results, depicted in Figure 5,
show that treatment of HT-29 cells with combretastatin A-4 and
the aforementioned derivatives dissolved in DMSO leads in fact
to various degrees of reduction in the transcription of hTERT and
Figure 4. Agarose gel profile of products resulting from RT-PCR amplification. The
total RNA of HT-29 cells previously treated with the appropriate combretastatin A-4
derivative was isolated, converted into cDNA, and amplified by PCR as described in
Section 6 (primers used for the RT-PCR are shown in Table 3). Gene expression of
VEGF and b-actin was quantified using the Image J program and normalized to that
of the housekeeping gene b-actin. At least three measurements were performed in
each case. Bars shown represent mean activations of VEGF gene expression and
error bars indicate standard errors of the mean. The statistical significance was
evaluated using one-sample t-tests (P <0.001).
Table 2
Inhibition of VEGF protein and of gene expression by combretastatin A-4 and some of
its O-alkyl derivativesa
Compound
% Protein inhibitionb
% Gene inhibitionc
CoA4
2b
2g
2i
2j
53
56
62
58
49
49
52
9
31
3
20
10
34
27
2k
2n
a
Concentrations used were below the IC50 values.
Values obtained by subtracting from 100 the percentages of VEGF secretion
b
values in Fig. 3.
c
Values obtained by subtracting from 100 the percentages of VEGF gene inhi-
bition values in Fig. 4.
in Section 1.12–16 Human telomerase contains an RNA component
(hTR) that serves as a template for the addition of the repeat nucle-
otide sequences and a protein subunit (hTERT) which catalyzes the
nucleotide polymerization process. In addition, there are other
associated protein factors, the role of which has not yet been com-
pletely elucidated. Human telomerase is regulated during develop-
ment and differentiation, mainly through transcriptional control of
the hTERT gene, the expression of which is restricted to cells that
exhibit telomerase activity. This indicates that hTERT is the rate
limiting factor of the enzyme complex.16a For the expression of
the hTERT gene, two transcriptional factors called c-Myc and Sp1,
among others, have been found to play an important role through
upregulation of the mRNA encoding the hTERT protein subunit of
telomerase.30 Thus, and as a preliminary study of the potential
anti-telomerase activity of combretastatin derivatives, we have
Figure 5. Agarose gel profile of products resulting from RT-PCR amplification. The
total RNA of HT-29 cells previously treated with the appropriate compound was
isolated, converted into cDNA, and amplified by PCR as described in Section 6
(primers used for the RT-PCR are shown in Table 3). Gene expression of hTERT, c-
myc and b-actin was quantified using the Image J program and normalized to that of
the housekeeping gene b-actin. At least three measurements were performed in
each case. Bars shown represent mean activations of hTERT and c-myc gene
expression and error bars indicate standard errors of the mean. The statistical
significance was evaluated using one-sample t-tests (P <0.001).
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S. Torijano-Gutiérrez et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
5
c-myc mRNA as compared with control cells. The most active deriv-
ative was found to be 2n, which proved able to reduce the expres-
sion of both genes to less than 40% of the control value. For the sake
of comparison, combretastatin A-4 caused a reduction to 74% of the
control value in the case of the hTERT gene and proved practically
inactive in the case of the c-myc gene.
Comparison of the results for the expression of the hTERT gene
with those of the c-myc gene reveals a good correlation between
them (Fig. 5). We may conclude therefore that the compounds un-
der study downregulate the expression of the hTERT gene by low-
ering the transcription of the c-myc gene.
otherwise indicated, with CDCl3 signals as internal reference. 13C
NMR signal multiplicities were determined with the DEPT pulse
sequence. Mass spectra were run in the electrospray (ESMS) mode
and, in some cases, in the electron impact mode (EIMS). Reactions
which required an inert atmosphere were carried out under dry N2
with flame-dried glassware. Commercial reagents were used as
received. Dichloromethane was freshly distilled from CaH2.
Column chromatography was performed on a silica gel column
(60–200 lm) with elution with the indicated solvent mixtures.
6.2. Reaction conditions
6.2.1. Styrene derivatives
5. Summary
Styrene derivatives 5–7 and 11–13 are commercially available.
Styrenes 8–10 were prepared as reported31 by means of Wittig
methylenation of the appropriate commercial benzaldehydes
(yields in the range 60–70%):
A series of O-alkyl derivatives of combretastatin A-4 has been
prepared and evaluated in relation to three types of biological
properties: cytotoxicity, antiangiogenesis and telomerase inhibi-
tion. One healthy (human embryonic kidney, HEK-293) and two
tumoral cell lines (human colon HT-29 and breast adenocarcinoma
MCF-7) were used for the assays.
Compound 8: oil; 1H NMR (500 MHz) d 7.54 (1H, dd, J = 7.7,
1.5 Hz), 7.26 (1H, td, J = 7.7, 1.5 Hz), 7.20–7.10 (2H, m), 7.04 (1H,
td, J = 7.5, 1 Hz), 5.80 (1H, dd, J = 17.7, 1.5 Hz), 5.33 (1H, dd,
J = 11, 1.5 Hz), 5.25 (2H, s), 3.53 (3H, s); 13C NMR (125 MHz) d
154.4, 127.6 (C), 131.5, 128.8, 126.4, 121.9, 114.8 (CH), 114.5,
94.7 (CH2), 56.0 (CH3).
No clear correlations are perceived between structure and
activity in the compounds under study. For instance, the strongest
cytotoxicities (lowest IC50 values) and the highest
a or b values are
Compound 9: oil; 1H NMR (500 MHz) d 7.26 (1H, t, J = 7.9 Hz),
7.12 (1H, br t, J ꢀ 2 Hz), 7.08 (1H, br d, J ꢀ 7.6), 6.97 (1H, ddd,
J = 7.9, 2.4, 1 Hz), 6.71 (1H, dd, J = 17.5, 10.8 Hz), 5.77 (1H, dd,
J = 17.5, 1 Hz), 5.27 (1H, dd, J = 10.8, 1 Hz), 5.21 (2H, s), 3.51 (3H,
s); 13C NMR (125 MHz) d 157.6, 139.2 (C), 136.7, 129.5, 120.1,
115.7, 114.0 (CH), 114.3, 94.5 (CH2), 56.0 (CH3).
found almost exclusively among the compounds containing the
(E)-arylalkene fragment, that is, within the 2f–2n group (Table 1).
However, the observed IC50 values do not bear a close relationship
with the
appreciable cytotoxicity towards both tumoral cell lines accompa-
nied by high and b values. In other cases (e.g., 2f, 2h, 2n), these
a/b coefficients. Only in the case of compound 2i was an
a
Compound 10: oil; 1H NMR (500 MHz) d 7.38 (2H, br d,
J ꢀ 8.5 Hz), 7.05 (2H, br d, J ꢀ 8.5 Hz), 6.71 (1H, dd, J = 17.6,
11 Hz), 5.77 (1H, d, J = 17.6 Hz), 5.21 (2H, s), 5.20 (1H, d,
J = 11 Hz), 3.52 (3H, s); 13C NMR (125 MHz) d 157.0, 131.6 (C),
136.2, 127.3 (ꢁ2), 116.3 (ꢁ2) (CH), 112.0, 94.4 (CH2), 55.9 (CH3).
desirable features were observed in only one of the two cell lines.
The inhibition of the VEGF production, as a measure of the anti-
angiogenic activity, did not show a very marked relation with the
structural type, either. Except for 2c and 2e, which were clearly
less active, all other compounds displayed comparable activities,
with 2g being the most active. The most outstanding aspect here
was that some of the compounds (2b, 2g and 2i) proved even more
active in this property than combretastatin A-4. Again, these be-
long to the 2f–2n group. From these, 2i also showed favourable
cytotoxicity indexes, as commented above.
The ability to inhibit the expression of the hTERT and c-myc
genes was also found in the investigated combretastatin A-4 deriv-
atives. The profile was, however, clearly different from that ob-
served in the two other biological properties. For example, the
strongest activity was found here in compound 2n, with com-
pounds 2b, 2g and 2i, which had favourable antiangiogenic fea-
tures, being much less active. Nonetheless, it is worth noting that
several of the compounds displayed a higher activity than combre-
tastatin A-4. Once again, the compounds of the 2f–2n group were
found more active than the simple O-alkyl derivatives.
In summary, some of the investigated combretastatin A-4 O-al-
kyl derivatives show an activity comparable or higher than that of
combretastatin A-4 itself in at least one of the three examined bio-
logical properties. While it seems that compounds containing the
(E)-arylalkene fragment (2f–2n) display in general stronger activi-
ties than the simple O-alkyl derivatives (2a–2e), no clear structure/
activity correlations were perceived, however, when comparing
the observed compound activities across the three biological prop-
erties. This point out the existence of marked differences between
the mechanisms responsible for their cytotoxicity.
6.2.2. Synthesis of bromides 3f–3n by means of cross metathesis
Representative example: a solution of bromide 4 (60
lL, 75 mg,
ca. 0.5 mmol) and styrene 6 (235 mg, 1.75 mmol) in dry, degassed
CH2Cl2 (50 mL) was treated with Ru-II catalyst (150 mg,
0.175 mmol). The reaction mixture was then stirred at reflux under
N2 for 24 h. Subsequently, the mixture was treated with DMSO
(600 l
L)32 and stirred overnight at room temperature. The reaction
mixture was then evaporated under reduced pressure, and the res-
idue was carefully chromatographed on silica gel (elution with
hexane–Et2O, 400:1).33 This gave 3g (55 mg, 43% based on 4) to-
gether with the stilbene derivative generated by homodimeriza-
tion of 6 (major product). In the other examples, yields were in
the range 35–50% except for bromides 3l–3n, which could not be
purified and were obtained only in admixture with variable per-
centages of the homodimerization products. The mixtures were
then used for the alkylation step.
Compound 3f: oil; 1H NMR (500 MHz) d 7.43 (1H, br d,
J ꢀ 7.8 Hz), 7.22 (1H, br t, J ꢀ 7.8 Hz), 6.94 (1H, t, J = 7.8 Hz), 6.88
(1H, br d, J ꢀ 7.8 Hz), 6.79 (1H, d, J = 16 Hz), 6.20 (1H, dt, J = 16,
7 Hz), 3.87 (3H, s), 3.48 (2H, t, J = 7 Hz), 2.42 (2H, br q, J ꢀ 7 Hz),
2.07 (2H, br quint, J ꢀ 7 Hz); 13C NMR (125 MHz) d 156.4, 126.4
(C), 129.2, 128.1, 126.5, 126.0, 120.6, 110.9 (CH), 33.2, 32.4, 31.8
(CH2), 55.5 (CH3); HR EIMS m/z (rel int.) 254.0351 (M+, 40), 239
(M+ꢂMe, 100). Calcd for C12H15 79BrO, 254.0306.
Compound 3g: oil; 1H NMR (500 MHz) d 7.23 (1H, t, J = 7.8 Hz),
6.96 (1H, br d, J ꢀ 7.8 Hz), 6.91 (1H, br t, J ꢀ 2 Hz), 6.79 (1H, dd,
J = 7.8, 2.5 Hz), 6.44 (1H, d, J = 16 Hz), 6.18 (1H, dt, J = 16, 7 Hz),
3.83 (3H, s), 3.47 (2H, t, J = 7 Hz), 2.40 (2H, br q, J ꢀ 7 Hz), 2.05
(2H, br quint, J ꢀ 7 Hz); 13C NMR (125 MHz) d 159.8, 138.9 (C),
131.2, 129.5, 128.8, 118.7, 112.8, 111.4 (CH), 33.1, 32.2, 31.2
(CH2), 55.2 (CH3); HR EIMS m/z (rel int.) 254.0336 (M+, 80), 175
6. Materials and methods
6.1. Chemistry: general procedures
General features. NMR spectra were recorded at 500 MHz (1H
NMR) and 125 MHz (13C NMR) in CDCl3 solution at 25 °C, if not
Please cite this article in press as: Torijano-Gutiérrez, S.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.064

6
S. Torijano-Gutiérrez et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
(M+ꢂBr, 100), 147 (M+ꢂBr–C2H4, 98). Calcd for C12H15 79BrO,
254.0306.
J = 12.1 Hz), 5.75-5.60 (2H, m), 4.30 (2H, br d, J ꢀ 5.8 Hz), 3.85
(3H, s), 3.84 (3H, s), 3.72 (6H, s), 1.70 (3H, br dd, J ꢀ 6.5, 1.5 Hz);
13C NMR (125 MHz) d 153.0 (ꢁ2), 148.7, 147.6, 137.2, 133.0,
129.8 (C), 130.8, 129.7, 128.7, 125.9, 122.0, 113.6, 111.1, 106.0
(ꢁ2) (CH), 69.5 (CH2), 60.9, 56.0 (ꢁ3), 17.7 (CH3); HR ESMS m/z
393.1677 (M+Na+). Calcd for C22H26NaO5, 393.1678.
Compound 3h: oil; 1H NMR (500 MHz) d 7.30 (2H, br d,
J ꢀ 8.5 Hz), 6.86 (2H, br d, J ꢀ 8.5 Hz), 6.41 (1H, d, J = 16 Hz), 6.04
(1H, dt, J = 16, 7 Hz), 3.82 (3H, s), 3.46 (2H, t, J = 7 Hz), 2.38 (2H,
br q, J ꢀ 7 Hz), 2.05 (2H, br quint, J ꢀ 7 Hz); 13C NMR (125 MHz)
d 158.9, 128.6 (C), 130.7 (ꢁ2), 127.1 (ꢁ2), 126.3, 114.0 (CH), 33.2,
32.4, 31.3 (CH2), 55.3 (CH3); HR EIMS m/z (rel int.) 254.0362 (M+,
45), 175 (M+ꢂBr, 12), 147 (M+ꢂBr–C2H4, 100). Calcd for C12H15
79BrO, 254.0306.
Compound 2c: solid, mp 83–84 °C; 1H NMR (500 MHz) d 7.35–
7.25 (5H, br m), 6.90–6.85 (2H, m), 6.80 (1H, d, J = 8.3 Hz), 6.51
(2H, s), 6.47 (1H, d, J = 12.2 Hz), 6.43 (1H, d, J = 12.2 Hz), 4.94
(2H, s), 3.87 (3H, s), 3.85 (3H, s), 3.71 (6H, s); 13C NMR
(125 MHz) d 153.0 (ꢁ2), 148.9, 147.7, 137.2, 136.9, 133.0, 129.8
(C), 133.1, 129.6, 128.4 (ꢁ2), 127.7, 127.2 (ꢁ2), 122.4, 114.5,
111.5, 106.0 (ꢁ2) (CH), 70.9 (CH2), 60.9, 56.0, 55.9 (ꢁ2) (CH3);
HR ESMS m/z 429.1674 (M+Na+). Calcd for C25H26NaO5, 429.1678.
Compound 2d: solid, mp 62–63 °C; 1H NMR (500 MHz) d 7.24
(2H, br d, J ꢀ 8.7 Hz), 6.95–6.85 (3H, m), 6.78 (2H, br d,
J = 8.7 Hz), 6.52 (2H, s), 6.47 (1H, d, J = 12.2 Hz), 6.43 (1H, d,
J = 12.2 Hz), 4.87 (2H, s), 3.85 (3H, s), 3.84 (3H, s), 3.81 (3H, s),
3.71 (6H, s); 13C NMR (125 MHz) d 159.4, 153.0 (ꢁ2), 149.0,
147.8, 137.2, 133.0, 129.9, 129.1 (C), 129.7, 129.0 (ꢁ2), 128.8,
122.4, 114.7, 113.9 (ꢁ2), 111.5, 106.0 (ꢁ2) (CH), 70.7 (CH2), 60.9,
55.9 (ꢁ3), 55.3 (CH3); HR ESMS m/z 459.1783 (M+Na+). Calcd for
C26H28NaO6, 459.1784.
Compound 3i: oil; 1H NMR (500 MHz)
d 7.45 (1H, br d,
J ꢀ 7.8 Hz), 7.19 (1H, br t, J ꢀ 7.8 Hz), 7.10 (1H, br d, J ꢀ 7.8 Hz),
6.98 (1H, br t, J ꢀ 7.8 Hz), 6.81 (1H, d, J = 16 Hz), 6.18 (1H, dt,
J = 16, 7 Hz), 5.23 (2H, s), 3.52 (3H, s), 3.49 (2H, t, J = 7 Hz), 2.42
(2H, br q, J ꢀ 7 Hz), 2.07 (2H, br quint, J ꢀ 7 Hz); 13C NMR
(125 MHz) d 154.1, 127.4 (C), 129.2, 128.1, 126.4, 125.8, 122.0,
115.0 (CH), 94.8, 33.2, 32.4, 31.7 (CH2), 56.1 (CH3); HR EIMS m/z
(rel int.) 284.0428 (M+, 60), 239 (M+ꢂCH2OMe, 100). Calcd for C13-
H1779BrO2, 284.0412.
Compound 3j: oil; 1H NMR (500 MHz) d 7.23 (1H, t, J = 7.8 Hz),
7.05 (1H, br t, J ꢀ 2 Hz), 7.01 (1H, br d, J ꢀ 7.8 Hz), 6.92 (1H, br
dd, J ꢀ 7.8, 2.2 Hz), 6.43 (1H, d, J = 15.8 Hz), 6.18 (1H, dt, J = 15.8,
6.8 Hz), 5.20 (2H, s), 3.50 (3H, s), 3.47 (2H, t, J = 6.8 Hz), 2.39 (2H,
br q, J ꢀ 6.8 Hz), 2.05 (2H, br quint, J ꢀ 6.8 Hz); 13C NMR
(125 MHz) d 157.5, 139.0 (C), 131.1, 129.5, 128.9, 119.8, 115.1,
113.7 (CH), 94.4, 33.0, 32.2, 31.2 (CH2), 55.9 (CH3); HR EIMS m/z
(rel int.) 284.0416 (M+, 100), 254 (M+ꢂCH2O, 36), 175 (M+ꢂCH2-
O–Br, 65). Calcd for C13H17 79BrO2, 284.0412.
Compound 2e: oil; 1H NMR (500 MHz) d 7.44 (2H, br d,
J ꢀ 8.4 Hz), 7.18 (2H, br d, J = 8.4 Hz), 6.88 (1H, dd, J = 8.3, 1.7 Hz),
6.83 (1H, d, J = 1.7 Hz), 6.80 (1H, d, J = 8.3 Hz), 6.50 (2H, s), 6.44
(2H, s), 4.87 (2H, s), 3.86 (3H, s), 3.85 (3H, s), 3.71 (6H, s); 13C
NMR (125 MHz) d 153.0 (ꢁ2), 149.0, 147.4, 137.2, 136.0, 133.0,
129.8, 121.6 (C), 131.5 (ꢁ2), 129.5, 128.8, 128.7 (ꢁ2), 122.8,
114.6, 111.5, 106.0 (ꢁ2) (CH), 70.2 (CH2), 60.9, 56.0 (ꢁ3) (CH3);
HR ESMS m/z 507.0782 (M+Na+). Calcd for C25H25 79BrNaO5,
507.0783.
Compound 3k: oil; 1H NMR (500 MHz) d 7.29 (2H, br d,
J ꢀ 8.5 Hz), 6.99 (2H, br d, J ꢀ 8.5 Hz), 6.41 (1H, d, J = 15.9 Hz),
6.05 (1H, dt, J = 15.9, 6.8 Hz), 5.18 (2H, s), 3.49 (3H, s), 3.46 (2H,
t, J = 6.8 Hz), 2.37 (2H, br q, J ꢀ 6.8 Hz), 2.04 (2H, br quint,
J ꢀ 6.8 Hz); 13C NMR (125 MHz) d 156.5, 131.5 (C), 130.6, 127.1
(ꢁ2), 126.8, 116.3 (ꢁ2) (CH), 94.5, 33.1, 32.3, 31.2 (CH2), 55.9
(CH3); HR EIMS m/z (rel int.) 284.0353 (M+, 85), 254 (M+ꢂCH2O,
76), 147 (C5H8Br+, 100). Calcd for C13H17 79BrO2, 284.0412.
Compound 2f: oil; 1H NMR (500 MHz) d 7.41 (1H, dd, J = 7.7,
1.5 Hz), 7.19 (1H, td, J = 7.7, 1.5 Hz), 6.91 (1H, td, J = 7.7, 1 Hz),
6.90–6.85 (3H, m), 6.78 (1H, d, J = 8 Hz), 6.74 (1H, br d, J ꢀ 15.8 Hz),
6.53 (2H, s), 6.50 (1H, d, J = 12.2 Hz), 6.45 (1H, d, J = 12.2 Hz), 6.22
(1H, dt, J = 15.8, 6.8 Hz), 3.90 (2H, t, J = 6.8 Hz), 3.86 (3H, s), 3.84
(6H, s), 3.70 (6H, s), 2.36 (2H, br q, J ꢀ 7 Hz), 1.94 (2H, br quint,
J ꢀ 7 Hz); 13C NMR (125 MHz) d 156.3, 153.0 (ꢁ2), 148.8, 148.1,
137.2, 133.0, 130.0, 126.8 (C), 130.4, 129.8, 128.8, 127.9, 126.5,
125.2, 122.0, 120.7, 113.9, 111.5, 110.8, 106.0 (ꢁ2) (CH), 68.4,
29.8, 28.8 (CH2), 60.9, 56.1, 56.0 (ꢁ2), 55.5 (CH3); HR ESMS m/z
513.2247 (M+Na+). Calcd for C30H34NaO6, 513.2253.
6.2.3. Synthesis of combretastatin A-4 derivatives 2a–2n
Representative example: A solution of 1a (63 mg, 0.2 mmol) in
dry DMF (3 mL) was treated under N2 in the dark at room temper-
ature with K2CO3 (70 mg, ca. 0.5 mmol) and stirred for 1 h. Subse-
quently, 3a (52 lL, 0.6 mmol) was added and the stirring was
continued for 24 h under the same conditions. The reaction mix-
ture was then poured into saturated aqueous NH4Cl and extracted
three times with Et2O (3 ꢁ 15 mL). The organic layer was dried
over anhydrous Na2SO4, filtered and evaporated under reduced
pressure. Column chromatography of the residue on silica gel (elu-
tion with hexane–EtOAc, 9:1) provided 2a (50 mg, 70%). In the
other cases, yields were in the range 40–70%. Bromides 3l–3n were
used in excess as the mixtures with the homodimerization prod-
ucts (see above). Caution: reaction, work-up and purification proce-
dures should be performed under minimization of exposure to light,
due to the ease of photoinduced Z/E isomerization in the combretast-
atin moiety.
Compound 2g: oil; 1H NMR (500 MHz) d 7.21 (1H, t, J = 7.9 Hz),
6.94 (1H, br d, J ꢀ 7.7 Hz), 6.90–6.85 (3H, m), 6.78 (1H, d,
J = 8.6 Hz), 6.76 (1H, dd, J = 8.3, 2.5 Hz), 6.53 (2H, s), 6.50 (1H,
d, J = 12 Hz), 6.45 (1H, d, J = 12 Hz), 6.38 (1H, br d, J ꢀ 15.8 Hz),
6.23 (1H, dt, J = 15.8, 6.8 Hz), 3.88 (2H, t, J = 6.7 Hz), 3.86 (3H,
s), 3.84 (3H, s), 3.82 (3H, s), 3.70 (6H, s), 2.34 (2H, br q, J ꢀ 7 Hz),
1.92 (2H, br quint, J ꢀ 7 Hz); 13C NMR (125 MHz) d 160.0, 153.0
(ꢁ2), 148.8, 148.0, 139.2, 137.2, 133.0, 130.0 (C), 130.4, 130.1,
129.7, 129.4, 128.8, 122.1, 118.7, 113.8, 112.5, 111.5 (ꢁ2),
106.0 (ꢁ2) (CH), 68.2, 29.4, 28.7 (CH2), 60.9, 56.0 (ꢁ3), 55.2
(CH3); HR ESMS m/z 513.2255 (M+Na+). Calcd for C30H34NaO6,
513.2253.
Compound 2a: solid, mp 107–108 °C; 1H NMR (500 MHz) d 6.86
(1H, dd, J = 8.3, 1.7 Hz), 6.84 (1H, d, J = 1.7 Hz), 6.79 (1H, d,
J = 8.3 Hz), 6.52 (2H, s), 6.50 (1H, d, J = 12.2 Hz), 6.46 (1H, d,
J = 12.2 Hz), 5.95 (1H, ddt. J = 17.2, 11.4, 5.5 Hz), 5.27 (1H, dq,
J = 17.2, 1.5 Hz), 5.20 (1H, dq, J = 11.4, 1.5 Hz), 4.40 (2H, dt,
J = 5.5, 1.5 Hz), 3.86 (3H, s), 3.84 (3H, s), 3.71 (6H, s); 13C NMR
(125 MHz) d 153.0 (ꢁ2), 148.7, 147.5, 137.2, 133.0, 129.8 (C),
133.1, 129.7, 128.8, 122.3, 113.9, 111.3, 106.0 (ꢁ2) (CH), 117.9,
69.7 (CH2), 60.9, 56.0 (ꢁ3) (CH3); HR ESMS m/z 379.1518
(M+Na+). Calcd for C21H24NaO5, 379.1521.
Compound 2h: oil; 1H NMR (500 MHz) d 7.28 (2H, br d,
J ꢀ 8.5 Hz), 6.90–6.85 (4H, m), 6.79 (1H, d, J = 8.2 Hz), 6.54 (2H,
s), 6.50 (1H, d, J = 12 Hz), 6.46 (1H, d, J = 12 Hz), 6.36 (1H, br d,
J ꢀ 15.8 Hz), 6.09 (1H, dt, J = 15.8, 6.8 Hz), 3.89 (2H, t, J = 6.7 Hz),
3.86 (3H, s), 3.85 (3H, s), 3.82 (3H, s), 3.71 (6H, s), 2.33 (2H, br q,
J ꢀ 7 Hz), 1.92 (2H, br quint, J ꢀ 7 Hz); 13C NMR (125 MHz) d
158.7, 153.0 (ꢁ2), 148.8, 148.0, 137.2, 133.0, 130.6, 130.0 (C),
129.8, 129.7, 128.7, 127.5, 127.1 (ꢁ2), 122.0, 113.9 (ꢁ2), 113.7,
111.4, 106.0 (ꢁ2) (CH), 68.2, 29.4, 28.8 (CH2), 60.9, 56.0 (ꢁ3),
55.2 (CH3); HR ESMS m/z 513.2256 (M+Na+). Calcd for C30H34NaO6,
513.2253.
Compound 2b: oil; 1H NMR (500 MHz) d 6.86 (2H, m), 6.74 (1H,
d, J = 8.5 Hz), 6.53 (2H, s), 6.50 (1H, d, J = 12.1 Hz), 6.44 (1H, d,
Please cite this article in press as: Torijano-Gutiérrez, S.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.064

S. Torijano-Gutiérrez et al. / Bioorg. Med. Chem. xxx (2013) xxx–xxx
7
Compound 2i: oil; 1H NMR (500 MHz) d 7.44 (1H, dd, J = 7.9,
1.8 Hz), 7.16 (1H, td, J = 7.9, 1.8 Hz), 7.07 (1H, dd, J = 8, 1 Hz),
6.97 (1H, td, J = 7.6, 1 Hz), 6.90–6.85 (2H, m), 6.78 (1H, d,
J = 8 Hz), 6.75 (1H, br d, J ꢀ 15.8 Hz), 6.53 (2H, s), 6.50 (1H, d,
J = 12 Hz), 6.45 (1H, d, J = 12 Hz), 6.22 (1H, dt, J = 15.8, 6.8 Hz),
5.20 (2H, s), 3.90 (2H, t, J = 6.8 Hz), 3.86 (3H, s), 3.84 (3H, s), 3.70
(6H, s), 3.48 (3H, s), 2.38 (2H, br q, J ꢀ 7 Hz), 1.94 (2H, br quint,
J ꢀ 7 Hz); 13C NMR (125 MHz) d 154.0, 153.0 (ꢁ2), 148.8, 148.1,
137.2, 133.0, 130.0, 127.7 (C), 130.5, 129.8, 128.8, 127.9, 126.4,
125.0, 122.0, 121.9, 114.9, 113.8, 111.5, 106.0 (ꢁ2) (CH), 94.8,
68.3, 29.8, 28.8 (CH2), 60.9, 56.1, 56.0, 55.9 (ꢁ2) (CH3); HR ESMS
m/z 543.2362 (M+Na+). Calcd for C31H36NaO7, 543.2359.
128.8, 127.5 (ꢁ2), 122.1, 113.7, 111.4, 106.0 (ꢁ2) (CH), 68.2,
29.4, 28.6 (CH2), 60.9, 56.1, 56.0 (ꢁ2) (CH3); HR ESMS m/z
561.1248 (M+Na+). Calcd for C29H31 79BrNaO5, 561.1253.
6.3. Biological procedures
6.3.1. Reagents and cell culture
Cell culture media were purchased from Gibco (Grand Island,
NY, USA) and Biowhittaker (Walkersville, MD, USA). Fetal bovine
serum (FBS) was a product of Harlan-Seralab (Belton, UK). Supple-
ments and other chemicals not listed in this section were obtained
from Sigma Chemicals Co. (St. Louis, MO, USA). Plastics for cell cul-
ture were supplied by NUNC (Roskilde, Denmark). Combretastatin
derivatives 2a–n (samples purified by HPLC) were dissolved in
DMSO at a concentration of 10 mg/mL and stored at ꢂ20 °C until
use.
Compound 2j: oil; 1H NMR (500 MHz) d 7.21 (1H, t, J = 7.8 Hz),
7.02 (1H, br s), 7.00 (1H, br d, J ꢀ 8 Hz), 6.90–6.85 (3H, m), 6.78
(1H, d, J = 8.4 Hz), 6.54 (2H, s), 6.50 (1H, d, J = 12 Hz), 6.46 (1H, d,
J = 12 Hz), 6.38 (1H, br d, J ꢀ 15.7 Hz), 6.23 (1H, dt, J = 15.7,
6.8 Hz), 5.19 (2H, s), 3.88 (2H, t, J = 6.6 Hz), 3.86 (3H, s), 3.84 (3H,
s), 3.70 (6H, s), 3.49 (3H, s), 2.34 (2H, br q, J ꢀ 7 Hz), 1.91 (2H, br
quint, J ꢀ 7 Hz); 13C NMR (125 MHz) d 157.5, 153.0 (ꢁ2), 148.8,
148.0, 139.2, 137.2, 133.0, 129.9 (C), 130.3, 130.2, 129.7, 129.4,
128.8, 122.0, 119.8, 114.8, 113.8, 113.7, 111.4, 106.0 (ꢁ2) (CH),
94.4, 68.2, 29.4, 28.7 (CH2), 60.8, 55.9 (x 4) (CH3); HR ESMS m/z
543.2359 (M+Na+). Calcd for C31H36NaO7, 543.2359.
Cell lines were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) containing glucose (1 g/L), glutamine (2 mM),
penicillin (50 IU/mL), streptomycin (50
lg/mL) and amphoterycin
(1.25 g/mL), supplemented with 10% FBS.
l
6.3.2. Cell proliferation assay
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT; Sigma Chemical Co., St. Louis, MO) dye reduction assay
in 96-well microplates was used, as previously described.34 Some
5 ꢁ 103 cells of HT-29, MCF-7 or HEK-293 cells in a total volume
Compound 2k: oil; 1H NMR (500 MHz) d 7.26 (2H, br d,
J ꢀ 8.5 Hz), 6.97 (2H, br d, J ꢀ 8.5 Hz), 6.90–6.85 (2H, m), 6.78
(1H, d, J = 8.2 Hz), 6.53 (2H, s), 6.50 (1H, d, J = 12.3 Hz), 6.45
(1H, d, J = 12.3 Hz), 6.36 (1H, br d, J ꢀ 15.7 Hz), 6.10 (1H, dt,
J = 15.7, 6.8 Hz), 5.17 (2H, s), 3.88 (2H, t, J = 6.8 Hz), 3.86 (3H,
s), 3.84 (3H, s), 3.70 (6H, s), 3.48 (3H, s), 2.32 (2H, br q, J ꢀ 7 Hz),
1.91 (2H, br quint, J ꢀ 7 Hz); 13C NMR (125 MHz) d 156.4, 153.0
(ꢁ2), 148.8, 148.0, 137.2, 133.0, 131.8, 130.0 (C), 129.8, 129.7,
128.8, 128.1, 127.1 (ꢁ2), 122.0, 116.3 (ꢁ2), 113.8, 111.4, 106.0
(ꢁ2) (CH), 94.5, 68.3, 29.4, 28.8 (CH2), 60.9, 56.0 (x 4) (CH3);
HR ESMS m/z 543.2360 (M+Na+). Calcd for C31H36NaO7,
543.2359.
of 100 lL of their respective growth media were incubated with se-
rial dilutions of the tested compounds. After 3 days of incubation
(37 °C, 5% CO2 in a humid atmosphere), 10 L of MTT (5 mg/ml
l
in PBS) were added to each well and the plate was incubated for
further 4 h (37 °C). The resulting formazan was dissolved in
150 lL of 0.04 N HCl/2-propanol and read at 550 nm. All determi-
nations were carried out in triplicate. The IC50 values in Table 1
mean the concentration of compound yielding a 50% of cell
survival.
Compound 2l: oil; 1H NMR (500 MHz) d 7.30 (2H, m), 6.98 (2H,
m), 6.90–6.85 (2H, m), 6.79 (1H, d, J = 8.3 Hz), 6.53 (2H, s), 6.50
(1H, d, J = 12.1 Hz), 6.45 (1H, d, J = 12.1 Hz), 6.37 (1H, br d,
J ꢀ 15.8 Hz), 6.14 (1H, dt, J = 15.8, 6.8 Hz), 3.88 (2H, t, J = 6.8 Hz),
3.86 (3H, s), 3.84 (3H, s), 3.70 (6H, s), 2.33 (2H, br q, J ꢀ 7 Hz),
1.91 (2H, br quint, J ꢀ 7 Hz); 13C NMR (125 MHz) d 162.9/161.0
6.3.3. RT-PCR analysis
HT-29 cells at 70–80% confluence were collected after serum
starvation for 24 h. Cells were incubated with combretastatin
derivatives in DMSO (see Figs. 4 and 5) for 48 h. Cells were
collected and the total cellular RNA from HT-29 cells was isolated
using Ambion RNA extraction Kit according to the manufacturer’s
instructions. The cDNA was synthesized by MMLV-RT with
[d, 1JC–F ꢀ 244 Hz], 153.0 (ꢁ2), 148.8, 148.0, 137.2, ꢀ133.9 [d, 4JC–F
-
ꢀ 3 Hz], 133.0, 129.9 (C), 129.7, 129.5, 129.3, 128.8, ꢀ127.4 [d,
3
JC–
2
F ꢀ 8 Hz] (ꢁ2), 122.1, 115.3/115.2 [d, JC–F ꢀ 21 Hz], (ꢁ2), 113.7,
111.4, 106.0 (ꢁ2) (CH), 68.2, 29.3, 28.7 (CH2), 60.9, 56.1, 56.0
(ꢁ2) (CH3); HR ESMS m/z 501.2052 (M+Na+). Calcd for C29H31-
FNaO5, 501.2053.
1–21 lg of extracted RNA and oligo(dT)15 according to the manu-
facturer’s instructions. Gene-specific PCR primers (see Table 3)
were then added for amplification. PCR products were analysed
by electrophoresis on 1.5% agarose gels and visualized by ethidium
bromide staining under UV transillumination. The sequences of
primers used in the RT-PCR are listed in Table 3. The PCR condi-
tions were as follows: VEGF at 94 °C for 30 s, at 58 °C for 1 min,
and at 72 °C for 1 min 50 s; and b-actin at 94 °C for 30 s, at 58 °C
for 50 s, and at 72 °C for 50 s; hTERT: 94 °C for 1 min, 57 °C for
1 min and 72 °C for 1 min 30 s; c-Myc: 94 °C for 30 s, 58 °C for
1 min, and 72 °C for 1 min 50 s.
Compound 2m: oil; 1H NMR (500 MHz) d 7.26 (4H, br s), 6.88
(1H, dd, J = 8.2, 1.7 Hz), 6.85 (1H, d, J = 1.7 Hz), 6.79 (1H, d,
J = 8.2 Hz), 6.53 (2H, s), 6.50 (1H, d, J = 12 Hz), 6.45 (1H, d,
J = 12 Hz), 6.36 (1H, br d, J ꢀ 15.8 Hz), 6.21 (1H, dt, J = 15.8,
6.8 Hz), 3.87 (2H, t, J = 6.8 Hz), 3.85 (3H, s), 3.84 (3H, s), 3.70 (6H,
s), 2.34 (2H, br q, J ꢀ 7 Hz), 1.91 (2H, br quint, J ꢀ 7 Hz); 13C NMR
(125 MHz) d 153.0 (ꢁ2), 148.8, 147.9, 137.2, 136.2, 133.0, 132.5,
129.9 (C), 130.5, 129.7, 129.3, 128.8, 128.5 (ꢁ2), 127.2 (ꢁ2),
122.1, 113.7, 111.4, 106.0 (ꢁ2) (CH), 68.2, 29.4, 28.6 (CH2), 60.9,
56.0 (ꢁ3) (CH3); HR ESMS m/z 517.1761 (M+Na+). Calcd for
Table 3
C
29H31 35ClNaO5, 517.1758.
Primers used and sizes of the PCR products
Compound 2n: oil; 1H NMR (500 MHz) d 7.41 (2H, br d,
VEGF
Sense: 50-CCTGATGAGATCGAGTACATCTT-30
Antisense: 50-ACCGCCTCGGCTTGTCAC-30
Sense: 50-CGGAAGAGTGTCTGGAGCAA-30
Antisense: 50-GGATGAAGCGGAGTCTGGA-30
Sense: 50-AAGTCCTGCGCCTCGCAA-30
Antisense: 50-GCTGTGGCCTCCAGCAGA-30
Sense: 50-TCATGAAGTGTGACGTTGACATC CGT-30
Antisense: 50-CGTAGAAGCATTTGCGGTGCAC GATG-30
379
145
249
287
J = 8.5 Hz), 7.20 (2H, br d, J = 8.5 Hz), 6.87 (1H, dd, J = 8.2, 1.8 Hz),
6.85 (1H, d, J = 1.8 Hz), 6.79 (1H, d, J = 8.2 Hz), 6.53 (2H, s), 6.50
(1H, d, J = 12 Hz), 6.45 (1H, d, J = 12 Hz), 6.35 (1H, br d, J ꢀ 15.8 Hz),
6.22 (1H, dt, J = 15.8, 6.8 Hz), 3.87 (2H, t, J = 6.8 Hz), 3.85 (3H, s),
3.84 (3H, s), 3.70 (6H, s), 2.33 (2H, br q, J ꢀ 7 Hz), 1.90 (2H, br quint,
J ꢀ 7 Hz); 13C NMR (125 MHz) d 153.0 (ꢁ2), 148.8, 147.9, 137.2,
136.6, 133.0, 131.5, 129.9 (C), 131.4 (ꢁ2), 130.7, 129.7, 129.4,
hTERT
c-Myc
b-Actin
Please cite this article in press as: Torijano-Gutiérrez, S.; et al. Bioorg. Med. Chem. (2013), http://dx.doi.org/10.1016/j.bmc.2013.09.064

8
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Financial support has been granted to M.C. by the Spanish
Government (Ministerio de Economía y Competitividad, projects
CTQ2008-02800 and CTQ2011-27560), by the Consellería d’Empre-
sa, Universitat i Ciencia de la Generalitat Valenciana (projects
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thanks the Generalitat Valenciana for a fellowship of the Santiago
Grisolía program. The authors further thank Rafael Pulido for
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Supplementary data
Supplementary data (graphical NMR spectra of compounds 2a–
2n) associated with this article can be found, in the online version,
at http://dx.doi.org/10.1016/j.bmc.2013.09.064.
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