Synthesis and biological evaluation of cyclic derivatives of combretastatin A-4 containing group 14 elements

Article abstract of DOI:10.1039/c8ob01148f

Several tricyclic compounds inspired by the structure of combretastatin A-4 and bearing group 14 elements have been synthesized by homocoupling lithiated aryl fragments followed by ring-closing metathesis. These tricyclic compounds and their diolefin prec

Full text of DOI:10.1039/c8ob01148f

View Article Online
View Journal
Organic &
Biomolecular
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: V. Blasco, J.
Murga, E. Falomir, M. Carda, S. Royo, A. C. Cuñat, J. Sanz and J. A. Marco, Org. Biomol. Chem., 2018,
DOI: 10.1039/C8OB01148F.
This is an Accepted Manuscript, which has been through the
Volume 14 Number
1 7 January 2016 Pages 1–372
Royal Society of Chemistry peer review process and has been
accepted for publication.
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-0520
COMMUNICATION
Takeharu Haino et al.
Solvent-induced emission of organogels based on tris(phenylisoxazolyl)
benzene
rsc.li/obc

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 1 of 13
View Article Online
DOI: 10.1039/C8OB01148F
Journal Name
ARTICLE
Synthesis and biological evaluation of cyclic derivatives of
combretastatin A-4 containing group 14 elements
Received 00th January 20,
Accepted 00th January 20
Víctor Blasco,a Juan Murga,b Eva Falomir, *b Miguel Carda,b Santiago Royo,b Ana C. Cuñat,*a Juan
F. Sanz-Cerveraa and J. Alberto Marcoa
DOI: 10.1039/x000000x
www.rsc.org/
Several tricyclic compounds inspired in the structure of combretastatin A-4 and bearing group 14
elements have been synthesized by homocoupling of lithiated aryl fragments followed by ring-
closing metathesis. These tricyclic compounds and their diolefin precursors were evaluated for their
antiproliferative action on the tumor cell lines HT-29, MCF-7, HeLa and A-549 and on the non-tumor
cell line HEK-293. In addition, their effects on the cell cycle were also measured. The tricyclic
compounds show antiproliferative activity similar to combretastatin A-4, even though they are not
so active in arresting cell cycle. However, some diolefin precursors are able to cause accumulation of
cells in the G2/M phase in a higher percentage than combretastatin A-4 itself. Inhibition of
endothelial tube formation and VEGFR-2 phosphorylation of some selected compounds are
comparable to that of combretastatin A-4, particularly those of tin-containing compounds 23c and
26c, whose actions exceed those of sorafenib, a clinically used VEGFR-2 inhibitor.
derivatives thereof.4 Developments in synthetic chemistry have
allowed the modification of natural products and the synthesis of
new chemical structures endowed with new and better
Introduction
Evolution of life on Earth has originated a myriad of organic
compounds from plants and microorganisms. These natural
products display a wide variety of structures, many of them
endowed with a plethora of organic functions resulting from
countless biosynthetic pathways. Since ancient times humankind
has used these compounds, particularly those extracted from
plants, as drugs to cure various diseases.1 Furthermore, natural
products have an ample range of binding groups that allow them to
interact within various biological targets in an optimal way.2 The
vast chemical diversity and novel mechanisms of action of natural
products explain the pivotal role they have been playing in many
drug development programmes.3
pharmacological properties. The incorporation of various types of
metal atoms into synthetic molecules, for instance, has allowed the
preparation of new pharmacologically valuable drugs, with cis-
platinum and its derivatives being archetypal examples.5 Our
interest on natural product analogues6 has led us to prepare several
of such analogues and to investigate their activity as
antiproliferative agents.7 Combretastatin A-4 (hereafter CA-4, see
structure I in Fig. 1), for instance, is a compound that belongs, like
the well-known resveratrol, to the stilbene class of natural
products.8 Several compounds of the combretastatin family have
acquired an outstanding status in the last years and found utility in
various pharmacological applications, most particularly as
anticancer agents.9 Preclinical and clinical developments over the
last decade have been rapidly accelerating for drugs such as I, most
particularly in the form of its more water-soluble phosphate
prodrug CA-4P (structure II, Fig. 1).10 These encouraging
developments have stimulated a variety of efforts devoted to the
synthesis and biological evaluation of numerous structurally
modified combretastatin derivatives and analogues.11
Cancer is a group of diseases characterized by the uncontrolled
growth and spread of abnormal cells. Cancer is caused by both
external and internal factors that may act together or in sequence
to initiate or promote carcinogenesis. Chemotherapy with cytotoxic
drugs is the main treatment for certain types of cancer. Among the
anti-cancer drugs approved in the period from 1940-2002,
approximately 54% were derived from natural products or
MeO
MeO
MeO
MeO
O
P
MeO
MeO
ONa
OH
O
ONa
OMe
OMe
I
II
This journal is © The Royal Society of Chemistry 20
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 2 of 13
View Article Online
DOI: 10.1039/C8OB01148F
ARTICLE
Journal Name
Fig. 1 Structures of combretastatin A-4 (I) and its phosphate
prodrug (II)
presence of potassium tert-butoxide to yield the cis stilbene
derivatives 6 and 7, respectively, with excellent stereoselectivity
(see Experimental for details on reaction conditions and yields).
Very recently, we have published several reports on the biological
properties of several CA-4 analogues with cytotoxic activity.12 One
of the potential drawbacks of I and derivatives is the fact that they
are prone to cis-trans isomerization induced by light or traces of
acid impurities. This is undesirable because the trans isomers are
practically devoid of biological activity.13 We thus conceived the
synthesis and biological evaluation of a family of CA-4 analogues
such as III (Fig. 2) in which the presence of an additional cycle
prevents the possibility of cis-trans isomerization.14 The atom Z may
belong in principle to any element of groups 14-16 in the periodic
system but we have selected for the initial phase several elements
of group 14 (Si, Ge, Sn).15 It is expected that the differences in C−Z
bond lengths, according to the nature of Z, will be reflected in
differences in the dihedral angle between the two benzene rings, a
feature which has been found to have an influence on the biological
activity.16
Scheme 2 Synthesis of dibromo derivatives 6-8. Abbreviations: TBS,
tert-butyldimethylsilyl; TBAF, tetra-n-butylammonium fluoride;
DIPEA, N,N-diisopropyl ethylamine.
The following step in the sequence proved disappointing. All
attempts to cyclize compounds 6 and 7 by means of bromine-
lithium interchange followed by treatment with the three
electrophilic reagents indicated below met with failure (Scheme 3).
In the case of compound 6 extensive decomposition took place
whereas in the case of 7, the product of reductive dehalogenation
was isolated in low yield. Its structure was confirmed by the fact
that desilylation yielded CA-4. A second side product isolated in low
yield seemed to be the result of lithiation followed by a retro-
Brook-type silyl migration.18 Conjecturing that the nature or else
the presence of the protecting group R might be the cause of the
failures, we also attempted the cyclization with the unprotected
compound 8, prepared as shown in Scheme 2. However, this
approach was not successful, as only CA-4 was isolated as a product
of reductive dehalogenation in low yield. While disappointing, these
results confirmed that at least bromine-lithium interchange had
actually taken place.
Fig. 2 General structure of tricyclic derivatives of CA-4
Results and discussion
Chemistry
The initial synthetic concept towards compounds of type III is that
depicted in Scheme 1. Thus, cyclic compounds such as III would be
obtained by reaction of the electrophilic reagent ZHal2 (Z = atom
from group 14, Hal = halogen) with the dilithium derivative IV to be
prepared in turn from dibromo derivative V. For the synthesis of the
latter compound we envisaged a Z-selective Wittig olefination, for
which there is a close precedent.17 The precursors would be the
ylide generated from phosphonium salt 1 and the protected
aldehyde 2 generated from the commercially available 3.
Scheme 3 Attempts to prepare tricyclic derivatives
After this lack of success, we reasoned that the failure in the
cyclization was possibly due to
a too slow reaction of the
electrophile ZHal2 with the C−Li bonds in the lithiated intermediate
IV (see Scheme 1). Since structurally close organolithium
compounds with a lower substitution degree in the rings reacted in
the expected way,17 these negative results may be due to both C−Li
bonds in IV being sterically hindered (ortho disubstitution). In view
of this, we decided to revert the order of formation of bonds of the
cyclic system. The two C−Z bonds would now be formed first by
means of an intermolecular process, followed by subsequent
creation of the C=C bond through an intramolecular olefin
metathesis. This new type of strategy, which has precedent,19 is
depicted in Scheme 4. Thus, III should be obtained by means of
ring-closing metathesis (RCM) of diolefin VI, to be in turn prepared
through sequential coupling of the electrophile ZHal2 with the two
Scheme 1 Retrosynthetic analysis
Scheme 2 shows the results of the synthetic work. Aldehyde 3
was protected as its allyl and tert-butyldimethylsilyl (TBS)
derivatives, 4 (R = allyl) and 5 (R = TBS), respectively. These were
then allowed to react with the known phosphonium salt 1 in the
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 3 of 13
View Article Online
DOI: 10.1039/C8OB01148F
Journal Name
ARTICLE
indicated organolithium reagents. The latter would be synthesized GeMe2, SnMe2) were obtained in two steps from 9 under the
by means of bromine-lithium interchange in bromoarenes VII and aforementioned reaction conditions.20 Subsequently, ring-closing
VIII.
olefin metathesis to combretastatin-type compounds 15 (Z = SiMe2,
GeMe2, SnMe2) was performed by means of heating at reflux a
solution of 14 and a second-generation Hoveyda-Grubbs ruthenium
catalyst21 in dry, deoxygenated toluene. The RCM step took place
with good yields for Z = SiMe2 and GeMe2 but with a low yield for Z
= SnMe2 (see Experimental for details on reaction conditions and
yields).
Scheme 4 Retrosynthetic analysis based on RCM
Compound 9 was synthesized by means of Wittig methylenation
of the commercially available 2-bromo-3,4,5-trimethoxy-
benzaldehyde. Likewise, 10 was prepared from aldehyde 3 through
sequential tritylation (Trt
(Scheme 5).
=
trityl) and Wittig methylenation
CHO
1: TrtCl, DMAP,
CH2Cl2, Et3N, RT
Scheme 7 Synthesis of tricyclic derivatives
MeO
MeO
Br
Br
+
Br
OH
I
2:
Ph PMe
OTrt
In order to expand the range of cyclic combretastatin-like
structures for biological investigation, we further performed the
same reaction sequence starting with three other aromatic,
commercially available aldehydes 16-18 (Scheme 8).
3
OMe
OMe
n-BuLi, THF
OMe
10
9
3
Scheme 5. Structure of 9 and synthesis of 10
CHO
+
Bromoarene was then converted into the corresponding
9
I
Ph3PMe
Br
Br
n-BuLi, THF
organolithium derivative 11 (n-BuLi, THF, −78°C), and the laꢀer was
treated with one equivalent of the appropriate electrophilic reagent
ZHal2 and stirred for 15 min. at the same temperature (Scheme 6).
Then the bath was allowed to reach room temperature and further
stirred for 12 h. Subsequently, the reaction mixture was treated
with one equivalent of the organolithium derivative 12 (generated
from 10) and further stirred for 12 h at room temperature.
RO
RO
OR´
OR´
16 R = R´= Me
17 R,R´= CH2
19 R = R´= Me
20 R,R´= CH2
+
CHO
I
Ph3PMe
Br
Br
n-BuLi, THF
F
F
18
21
1: nBuLi, THF
78 C
MeO
MeO
OMe
Ru cat.
MeO
OMe
OMe
Z
19
Z
OMe
tol,
MeO
2: ZCl2 (0.5 eq),
78 C RT
22a Z = SiMe2
22b Z = GeMe2
22c Z = SnMe2
25a Z = SiMe2
25b Z = GeMe2
Scheme 6 Attempts to prepare 13
1: nBuLi, THF
O
O
O
O
O
78 C
Ru cat.
tol,
Unfortunately, none of the desired heterocoupling compound 13
was obtained. Products of reductive dehalogenation were isolated
in low yield, together with products of homocoupling (see below).
We then tried an inversion of the order of the steps: organolithium
12 was allowed to react with the electrophile ZHal2 followed by
addition of 11. The same lack of success was observed. As
commented, the reaction mixtures above (Scheme 6) were shown
to contain products of homocoupling. In view of this, we considered
the possibility of preparing symmetric compounds such as 14
(Scheme 7), which still display the pharmacologically important vic-
trimethoxyphenyl fragment. Indeed, compounds 14 (Z = SiMe2,
20
Z
Z
O
O
O
2: ZCl2 (0.5 eq),
23a Z = SiMe2
23b Z = GeMe2
23c Z = SnMe2
26a Z = SiMe2
26b Z = GeMe2
26c Z = SnMe2
78 C
RT
1: nBuLi, THF
78 C
F
F
F
F
Ru cat.
tol,
21
Z
Z
2: ZCl2 (0.5 eq),
78 C
24a Z = SiMe2
24b Z = GeMe2
24c Z = SnMe2
27a Z = SiMe2
27b Z = GeMe2
27c Z = SnMe2
RT
Scheme 8 Synthesis of tricyclic derivatives 25-27
This journal is © The Royal Society of Chemistry 20
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 4 of 13
View Article Online
DOI: 10.1039/C8OB01148F
ARTICLE
Journal Name
Thus, the cyclic, combretastatin-like compounds 25-27 were
obtained (see Experimental for details on reaction conditions and
yields). As in the previous case, the RCM step gave good yields
when Z = SiMe2, GeMe2 but low yields when Z = SnMe2 (indeed, 0%
for 25c). In these cases, extrusion of the tin fragment took place
with formation of phenantrene derivatives. This behavior has
precedent in similar systems with group 15 elements.19c
these rigid compounds, although deeper studies would be
necessary to confirm this hypothesis.
Biological results
Effect on the inhibition of cell proliferation
The inhibition of cell proliferation of CA-4 and its tricyclic
derivatives was measured by means of their IC50 values towards the
tumor cell lines HT-29 (human colon adenocarcinoma), MCF-7
(breast adenocarcinoma), HeLa (human epithelioid cervix
carcinoma), A-549 (human lung adenocarcinoma) and in non-tumor
cell line HEK-293 (human embryonic kidney). In addition to the
compounds indicated in schemes 7 and 8, the acyclic geometrical
isomers 28 and 2922 drawn in Scheme 9 were also evaluated for
comparison purposes.
Scheme 9 Structures of (Z) and (E)-1,2-bis(3,4,5-trimethoxyphenyl)
ethylene (28 and 29, respectively)
IC50 values are presented in Table 1 along with the calculated
selectivity indexes (SI) obtained by dividing the IC50 values of the
non-tumor cell line (HEK-293) by those of the corresponding tumor
cell line. The higher the SI index of the compound, the higher its
therapeutic safety margin.
Most of the cyclic derivatives show antiproliferative activity in the
low micromolar range although they are not as active as CA-4. The
most active compounds are 15a-c, 25a and 26a-c, particularly on
the HT-29 and MCF-7 lines. Fluorinated compounds 27a-c exhibited
much lower antiproliferative actions. As regards selectivity indexes,
the best compounds are 15b, 26c and 27b with SI values well above
1. Furthermore, when the IC50 values of 28 and 29 are compared, it
is observed that the trans 29 isomer is much less active than the cis
isomer 28. This confirms previous observations that
a cis
configuration is needed in order to achieve noticeable
a
antiproliferative acivity.14 Besides, IC50 values of the acyclic
compound 28 are quite similar to those of tricyclic analogues 15a-c,
which means that the presence of the bridging atom does not
perturb the antiproliferative action.
The acyclic precursor compounds 22a-c, 23a-c and 24a-c were
also evaluated. Their IC50 values were also in the ꢀM range,
although they were slightly higher than those of their tricyclic
analogues and their SI values were lower than their tricyclic
derivatives. In this sense, it is worth mentioning that compound 23c
is the one with the highest selectivity index. The higher
antiproliferative activity of the tricyclic derivatives could be due to a
more favourable relative spatial orientation of the benzene rings in
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 5 of 13
View Article Online
DOI: 10.1039/C8OB01148F
Journal Name
ARTICLE
Table 1. IC50 (µM) values and SI indexes for combretastatin A-4, acyclic and cyclic derivatives.a
b
c
d
e
Comp.
CA-4
14a
14b
14c
15a
15b
15c
22a
22b
22c
23a
23b
23c
24a
24b
24c
25a
25b
26a
26b
26c
27a
27b
27c
28
HT-29
4.2 ± 0.5
7 ± 2
MCF-7
1.0 ± 0.2
6.2 ± 1
6.0 ± 0.8
7.0 ± 0.3
11.3 ± 0.2
9.15 ± 0.05
4.75 ± 0.02
43 ± 2
HeLa
2.5 ± 7
A-549
0.43 ± 0.004
7.4 ± 0.9
8 ± 2
HEK-293
25 ± 3
SIA
SIB
SIC
SID
6
25
10
58
3.9 ±0.7
4.7 ± 0.9
5.6 ± 0.4
6.6 ± 0.8
80 ± 15
2.0 ± 0.9
29 ± 8
1.4 ± 0.2
0.42 ± 0.05
0.26 ± 0.03
14 ± 2
0.2
0.2
0.3
0.2
11 ± 2
0.04 0.07 0.09 0.09
0.08 0.04 0.06 0.03
3.2 ± 0.8
8.3 ± 0.7
12.5 ± 0.8
2.2 ± 0.4
13 ± 2
8.6 ± 0.8
7.2 ± 0.3
22.7 ± 0.5
4.49 ± 0.02
57 ± 2
4.2
2.5
0.8
5
5.7
1.2
2
0.6
2.8
1.3
2.2
0.7
1.3
0.4
0.8
48
2.3
1.2
0.2
1.1
0.05
2.6
0.1
0.07
1
52 ± 9
5.6 ± 0.3
64 ± 2
1.5
0.07
0.7
0.07
0.8
6.8
2.3
1
23 ± 5
50 ± 8
5.5 ± 0.9
20 ± 4
76 ± 2
3.8 ± 0.3
26 ± 4
0.2
0.7
0.4
0.8
2.7
1
37 ± 2
35 ± 3
10 ± 2
5.6 ± 0.9
8 ± 1.5
27 ± 2
36 ± 2
5.5 ± 0.8
7.6 ± 0.7
48 ± 4
19 ± 2
2.5 ± 0.3
6.5 ± 1.0
>100
7.0 ± 1.2
68 ± 5
>100
>100
>100
44 ± 3
52 ± 6
74 ± 5
>100
2
1.3
1.2
1.7
0.4
0.4
0.06
45 ± 2
82 ± 2
70 ± 9
68 ± 4
85 ± 9
2
1.2
0.4
0.1
0.8
1
57 ± 5
18 ± 1
>100
21 ± 1
36 ± 5
0.6
0.9
0.4
0.8
0.5
0.7
0.4
1.7
0.9
0.3
-
2
7.5 ± 0.7
12 ± 2
3.2 ± 0.6
32 ± 5
5.0 ± 0.8
89 ± 2
26 ± 3
6.4 ± 0.2
11 ± 2
0.3
0.8
0.2
0.7
4.4
0.8
2
25 ± 6
8.4 ± 0.3
8.5 ± 0.6
15 ± 3
32 ± 10
6 ± 2
6.6 ± 0.8
80 ± 15
2.0 ± 0.9
55 ± 10
62 ± 8
>100
6.4 ± 0.5
4.2 ± 0.3
11 ± 2
54 ± 3
0.05 0.08
2.5 ± 0.2
57 ± 3
5.1 ± 0.2
>100
5.5
0.8
1.5
0.5
1.3
-
2.1
0.4
0.9
0.4
0.43
-
>100
45 ± 2
54 ± 9
45 ± 4
>100
92 ± 7
35 ± 2
53 ± 2
66 ± 4
76 ± 7
32 ± 5
0.6
1.1
-
8.0 ± 0.9
2.2 ± 0.2
1.82 ± 0,04
5.6 ± 0.5
2.4 ± 0.3
29
>100
>100
>100
21.5 ± 0.8
>100
aIC50 values are expressed as the compound concentration (ꢀM) that inhibits the cell growth by 50%. Data are the average (±SD) of three experiments. bSIA =
IC50(HEK-293)/IC50(HT-29). cSIB = IC50(HEK-293)/IC50(MCF-7). dSIC= IC50 (HEK-293)/IC50 (HeLa). eSID= IC50 (HEK-293)/IC50 (A-549).
This journal is © The Royal Society of Chemistry 20
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 6 of 13
View Article Online
DOI: 10.1039/C8OB01148F
Journal Name
ARTICLE
Effect on the cell cycle
suggests
a certain degree of antimitotic activity of these
compounds.
The effects of compounds on the cell cycle distribution were
evaluated in A-549 cells. Thus, cells were incubated for 20 h in the
presence of each compound at concentrations half of their IC50
values. Then the ADN content was measured by flow cytometry
Effect on microtubules
CA-4 is able to arrest the cell cycle because it interferes with the
(see experimental section). Along with the tricyclic compounds and formation of the microtubule cytoskeleton. In order to check
compounds 28 and 29, diolefin compounds 23a-c were also whether compounds 23a-c and 26a-c exhibited the same mode of
evaluated. These compounds were specifically selected for action as CA4, an immunofluorescence assay on A-549 cell line was
evaluation because they caused the formation of rounded cells.
performed. Thus, A549 cells were incubated for 16 h in the
presence of CA-4 and compounds 23a-c and 26a-c. Figure 3 depicts
the images of the effect caused by these compounds at their
minimum concentration. As a control assay, in which A-549 cells
Table 2. Cell cycle distribution.a
Comp.
Conc.
SubG0
G0
G1/S
G2/M
were treated with DMSO (Fig. 3a),
a normal filamentous
Control
CA-4
14a
14b
14c
15a
15b
15c
22a
22b
22c
23a
23b
23c
24a
24b
24c
25a
25b
26a
26b
26c
27a
27b
27c
28
---
5
15
2
69
21
87
86
70
88
89
80
68
71
44
24
23
20
84
74
76
80
71
35
47
26
65
72
80
39
72
12
22
5
13
43
4
microtubule network was observed, with microtubules extending
from the central regions of the cell to the periphery. It can be
appreciated that in the presence of a 50 nM dosis of CA-4 (Fig. 3b),
tubulin appears aggregated and nuclei are compressed and
fragmented, which is characteristic of cell division disruption.23
50 nM
5 ꢀM
5 ꢀM
2
6
6
5 ꢀM
6
10
6
12
4
5 ꢀM
2
5 ꢀM
2
6
3
5 ꢀM
10
13
10
54
5
7
3
50 ꢀM
50 ꢀM
5 ꢀM
10
8
7
9
5
3
5 ꢀM
10
11
12
2
61
60
65
4
50 ꢀM
50 ꢀM
50 ꢀM
50 ꢀM
5 ꢀM
5
2
3
8
2
12
10
6
9
4
25 ꢀM
25 ꢀM
50 ꢀM
50 ꢀM
5 ꢀM
50 ꢀM
50 ꢀM
50 ꢀM
5 ꢀM
5
9
13
9
7
8
10
12
7
46
30
49
19
5
11
11
3
12
4
19
7
Fig. 3. Effects on the microtubule network. A-549 cells were treated
for 16 hours and processed for immunofluorescence microscopy:
(a) DMSO; (b) 50 nM CA-4; (c) 25 µM of 23c; (d) 5 µM of 26c.
6
7
32
15
14
8
14
4
29
5 ꢀM
aAt least three measurements were performed in each case.
Compound 23c caused depolymerization and solubilization of
the microtubule cytoskeleton at concentrations around 25
ꢀM (Fig.
The results shown in Table
2 indicate that the tricyclic
3c). Furthermore, cells treated with a 5 M concentration of
ꢀ
analogues are unable to accumulate cells on G2/M phase to a
significant extent. In other words, these compounds are practically
devoid of antimitotic action. However, it is worth noting that the
diolefinic precursors 23a-c show a noticeable percentage of cells in
G2/M phase, with larger values than CA-4. Even though this effect is
exerted at concentrations >100 times greater than that of CA-4, this
compound 26c (Fig. 3d) showed disorganized interphase
microtubules. These morphological changes of microtubules
indicate that compounds of this structural type are able to disrupt
the microtubule morphology in a similar manner to CA-4.
Effect on endothelial cells
This journal is © The Royal Society of Chemistry 20
J. Name., 2013, 00, 1-3 | 6
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 7 of 13
View Article Online
DOI: 10.1039/C8OB01148F
Journal Name
ARTICLE
It has recently been shown that CA-4 might exhibit anti-
angiogenic properties by interfering the VEGF/VEGFR-2 signalling
pathway in endothelial cells.24 Thus, we studied the effect of our
synthetic derivatives on the proliferation of human microvascular
endothelial (HMEC-1) cells by MTT assay. For this study we selected
derivatives 23a-c and 26a-c, which were the ones that accumulated
more cells in the G2/M phase. CA-4 and Sorafenib,25 a clinically
used VEGFR-2 inhibitor,26 were employed as positive controls. Table
3 shows the IC50 values obtained for the tested compounds.
Table 3. IC50 values on HMEC-1 cells and minimum active
concentration.a
Minimum active
concentration (µM)
IC50 ± SD
HMEC-1 (µM)
Compound
CA-4
3.4 ± 0.4
34 ± 3
0.003
10
Sorafenib
23a
23b
23c
26a
26b
26c
35 ± 2
32 ± 9
12 ± 7
39 ± 9
27 ± 8
15 ± 4
5
5
Fig. 3. Effect of compound 23c at 5 µM (a), 0.5 µM (b), 0.05 µM
(c) and 0.025 µM (d) concentrations.
0.025
5
1
Inhibition of VEGFR-2 kinase activity
0.25
The effect of the selected compounds exerted over VEGFR-2, a
kinase receptor that plays an important role in the tumour
angiogenic process, was also evaluated. In this assay, HMEC-1 cells
were treated with the selected compounds for 2 hours, then VEGF
(50 ng/mL) was added to the cell media and 30 minutes later, cells
were lysed. Finally, phospho-VEGFR-2 was quantified from the
lysates by ELISA analysis. The percentage of phospho-VEGFR-2 is
expressed for each compound referred to control, which
corresponds to 100 %.
aAt least three measurements were performed in each case.
The data of Table 3 indicate a decrease of IC50 values for HMEC-
1 cells with the atomic number of the heteroatom present in the
compound. Thus, the tin compounds 23c and 26c exhibit half the
IC50 value of the silicon derivatives 23a and 26a.
Table 3 also shows the Minimum Active Concentration (MAC) at
which the selected compounds begin to inhibit VEGF-induced
endothelial tube formation. In order to measure this effect, HMEC-1
cells were seeded on Matrigel and the selected derivatives 23a-c
and 26a-c, along with CA-4 and sorafenib, were immediately added
Table 4. Detection of p-VEGFR-2 by ELISA assay.
Compound
CA-4
Conc. (µM)
% p-VEGFR-2
74 ± 8
at seriate concentrations ranging from 50 ꢀM to 10 nM. The effect
of the compounds on the endothelial tube formation was evaluated
5
after 24 h of treatment. As an example, the photos of the inhibitory
Sorafenib
25
60 ± 6
action of compound 23c (MAC 0.025
The most active compound is CA-4 which starts to inhibit
endothelial tube formation at 0.003 M. In this biological action all
ꢀM) are presented in Fig. 3.
23a
23b
23c
26a
26b
26c
25
25
25
25
25
25
64 ± 3
61 ± 3
52 ± 4
84 ± 6
82 ± 2
70 ± 4
ꢀ
synthetic compounds are more active than sorafenib itself. As
above, the tin derivatives 17c and 24c proved the most active.
It can be deduced from Table 4 that acyclic compounds 23a-c
are more active than cyclic derivatives 26a-c in causing the
inhibition of the kinase action of VEGFR-2 in endothelial cells.
Moreover, compounds 23a-c exert an inhibitory effect similar to
that of sorafenib or the lead compound, CA-4. Again, the action of
the compounds depends on the heteroatom they contain, the tin-
This journal is © The Royal Society of Chemistry 20
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 8 of 13
View Article Online
DOI: 10.1039/C8OB01148F
ARTICLE
Journal Name
containing compounds being a little more active than the rest of the mixture was extracted with CH2Cl2. The organic layer was
synthetic compounds.
washed with brine and then dried over anhydrous MgSO4.
Removal of volatiles under reduced pressure gave
4,
1
sufficiently pure for further use: oil; H NMR (300 MHz, CDCl3)
10.16 (1H, s), 7.41 (1H, s), 7.05 (1H, s), 6.05 (1H, ddt, J = 17.3,
Conclusions
δ
10.5, 5.5 Hz), 5.43 (1H, ddt, J = 17.3, 1.5, 1.5 Hz), 5.32 (1H, ddt,
A range of 23 compounds containing silicon, germanium
and tin have been synthesized and biologically evaluated as
regards their antiproliferative action, their effect on cell cycle,
and their effects on endothelial tube formation and inhibition
of VEGFR-2 kinase activity. As compounds with structures
inspired by that of combretastatin A-4, their biological actions
have been compared with those of the natural product.
J = 10.5, 1.5, 1.5 Hz), 4.63 (2H, dt, J = 5.5, 1.5 Hz), 3.95 (3H, s);
13C NMR (75 MHz, CDCl3)
δ 155.0, 147.9, 126.6, 120.6 (C),
190.9, 132.3, 115.8, 112.2 (CH), 119.0, 70.0 (CH2), 56.6 (CH3).
2-Bromo-5-(tert-butyldimethylsilyloxy)-4-methoxybenzaldehyde
(5). Aldehyde 3 (231 mg, 1 mmol) was dissolved under Ar in
dry THF (6 mL), cooled to 0°C and treated with TBSCl (226 mg,
1.5 mmol) and imidazole (204 mg, 3 mmol). The mixture was
then stirred for 16 h at room temperature. After this time,
water (10 mL) was added and the reaction mixture was
extracted with EtOAc. The organic layer was washed with satd
NaHCO3, then with brine and finally dried over anhydrous
MgSO4. Removal of volatiles under reduced pressure gave an
oily residue which was chromatographed on silica gel (elution
In the case of the tricyclic analogues 15, 25, 26 and 27,
where the third ring was expected to rigidify the structure, it
has been found that their antiproliferative action and their
effect on the cell cycle are weaker than that of CA-4. Thus, and
in contrast with our expectations, the introduction of the third
ring containing the heteroelement (Si, Ge, Sn) does not cause
the desired increase of the antiproliferative action. However,
their inhibition of endothelial tube formation and their
inhibition of VEGFR-2 phosphorylation are comparable to that
of CA-4, particularly in the case of the tin-containing
compounds 23c and 26c, whose actions exceed those of
sorafenib, a clinically used VEGFR-2 inhibitor.
with hexane-EtOAc 4:1). This gave
further use (259 mg, 75%): oil; 1H NMR (300 MHz, CDCl3)
10.14 (1H, s), 7.39 (1H, s), 7.03 (1H, s), 3.88 (3H, s), 0.98 (9H,
s), 0.16 (6H, s); 13C NMR (75 MHz, CDCl3)
156.9, 145.1, 126.9,
5, sufficiently pure for
δ
δ
120.5, 18.5 (C), 190.9, 120.6, 116.1 (CH), 56.1, 25.7 (x 3), −4.5
(x 2) (CH3).
The presence of compounds containing elements such as
tin immediately raises questions related to their potential
toxicity. While we have not performed toxicity studies on
these tricyclic analogues of CA-4, it is worth mentioning that
among the non-transition metal derivatives, more
organotin(IV) compounds have undergone testing as potential
anticancer agents than any other single group of compounds.27
The design of improved organotin(IV) antitumor agents
(Z)-1-(5-Allyloxy-2-bromo-4-methoxystyryl)-2-bromo-3,4,5-
trimethoxybenzene
(
6). A solution of triphenyl (2-bromo-
3,4,5-trimethoxybenzyl) phosphonium bromide
1
(300 mg, 0.5
mmol) in dry THF (8 mL) was cooled to 0°C and treated under
Ar with KOtBu (62 mg, 0.55 mmol). The mixture is then stirred
for 30 min. at 0 °C. A solution of the appropriate aldehyde
4
(0.4 mmol) in dry THF (8 mL) is then added dropwise, and the
mixture is stirred at room temperature for 16 h. After this
time, water (30 mL) was added and the reaction mixture was
extracted with EtOAc. The organic layer was washed with brine
and finally dried over anhydrous MgSO4. Removal of volatiles
under reduced pressure gave an oily residue which was
chromatographed on silica gel (elution with hexane-EtOAc
therefore still occupies
chemotherapy.27a
a significant place in cancer
Experimental
General features
9:1). The desired olefination product was obtained as an off-
1
white solid in 93% yield: oil; H NMR (300 MHz, CDCl3)
NMR spectra (300 MHz for 1H, 75 MHz for 13C and 282 MHz for
19F) were measured at 25°C. The signals of the deuterated
solvent (CDCl3) were taken as the reference for 1H and 13C
spectra. For 19F spectra (measured under 1H decoupling), δ
values were referenced to CFCl3 (δ = 0). 13C NMR signal
multiplicities were determined with the DEPT pulse sequence.
Mass spectra were run in the electrospray (ESMS) mode.
Experiments which required an inert atmosphere were carried
out under dry Ar in a flame-dried glassware. Commercially
available reagents were used as received. Acronyms are
defined in the synthetic schemes.
δ 7.01
(1H, s), 6.68 (1H, d, J = 11.8 Hz), 6.59 (1H, d, J = 11.8 Hz), 6.53
(1H, s), 6.40 (1H, s), 5.76 (1H, ddt, J = 17.3, 10.5, 5.5 Hz), 5.16
(1H, ddt, J = 17.3, 1.5, 1.5 Hz), 5.11 (1H, ddt, J = 10.5, 1.5, 1.5
Hz), 4.18 (2H, dt, J = 5.5, 1.5 Hz), 3.89 (3H, s), 3.84 (3H, s), 3.83
(3H, s), 3.49 (3H, s); 13C NMR (75 MHz, CDCl3)
δ 152.5, 151.1,
149.4, 146.9, 142.4, 132.9, 128.9, 114.8, 110.3 (C), 132.5,
130.3, 130.1, 115.3, 115.2, 109.8 (CH), 118.4, 69.8 (CH2), 61.2,
61.1, 56.2, 56.0 (CH3).
(Z)-[4-Bromo-5-(2-bromo-3,4,5-trimethoxystyryl)-2-methoxy-
phenoxy](tert-butyl)dimethylsilane
(
7
). Prepared as above in
and aldehyde : off-white
δ 7.01 (1H, s), 6.65 (1H, d, J =
5-(Allyloxy)-2-bromo-4-methoxybenzaldehyde (4). Aldehyde 3
82% yield from phosphonium salt
1
gum; H NMR (300 MHz, CDCl3)
1
5
(231 mg, 1 mmol) was dissolved under Ar in dry acetonitrile (8
mL) and treated with anhydrous K2CO3 (207 mg, 1.5 mmol).
11.8 Hz), 6.62 (1H, d, J = 11.8 Hz), 6.52 (1H, s), 6.39 (1H, s),
Then allyl bromide (105 ꢀL, 1.2 mmol) was added dropwise.
The mixture was then stirred for 24 h at room temperature.
3.89 (3H, s), 3.85 (3H, s), 3.78 (3H, s), 3.51 (3H, s), 0.84 (9H, s),
−0.08 (6H, s); 13C NMR (75 MHz, CDCl3)
δ 152.4, 151.3, 151.1,
After this time, water (10 mL) was added and the reaction
8 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 9 of 13
View Article Online
DOI: 10.1039/C8OB01148F
Journal Name
ARTICLE
144.2, 142.4, 133.0, 129.4, 115.2, 110.3, 18.4 (C), 130.3 (x 2), 127.7 (x 6), 127.5 (x 3), 119.9, 115.8 (CH), 114.4 (CH2), 56.1
122.7, 115.8, 109.7 (CH), 61.2, 61.1, 56.0, 55.9, 25.6 (x 3), −4.8 (CH3).
(x 2) (CH3).
Dimethyl bis(2,3,4-trimethoxy-6-vinylphenyl)silane
Compound (2.1 mmol) was dissolved under Ar in dry THF (7
7 (118 mg, 0.2 mmol) was dissolved mL) and cooled to −78°C. A 2.5M solution of n-BuLi in hexane
(14a).
(Z)-4-Bromo-5-(2-bromo-3,4,5-trimethoxystyryl)-2-methoxy-
phenol ). Compound
9
(
8
under Ar in dry THF (6 mL) and cooled to 0°C. A solution of 1M (0.84 mL, 2.1 mmol) was added dropwise and the mixture was
TBAF in THF (0.3 mL, 0.3 mmol) was slowly added dropwise. stirred at the same temperature for 30 min. Then, Me2SiCl2 (1
The mixture is then stirred at room temperature for 1 h. After mmol) dissolved in dry THF (2 mL) was added dropwise with
this time, satd aq NaHCO3 (10 mL) was added and the reaction stirring, and the mixture was allowed to reach room
mixture was extracted with EtOAc. The organic layer was temperature. The stirring was maintained for 12 h. After this
washed three times with brine and finally dried over time, satd aq NH4Cl (5 mL) was added and the reaction mixture
anhydrous MgSO4. Compound
8 was obtained in almost was extracted with CH2Cl2. The organic layer was washed three
quantitative yield, sufficiently pure for further use: off-white times with brine and finally dried over anhydrous MgSO4.
1
gum; H NMR (300 MHz, CDCl3)
δ 7.02 (1H, s), 6.68 (1H, d, J = Removal of volatiles under reduced pressure gave an oily
11.8 Hz), 6.67 (1H, s), 6.62 (1H, d, J = 11.8 Hz), 6.44 (1H, s), residue which was chromatographed on silica gel (elution with
1
3.90 (3H, s), 3.86 (6H, s), 3.48 (3H, s) (OH proton not detected); hexane-Et2O 95:5) to yield 14a (40%): off-white gum; H NMR
13C NMR (75 MHz, CDCl3)
δ
132.3, 130.1, 113.4, 110.7 (C), 130.2, 130.0, 116.4, 114.6, 5.40 (2H, dd, J = 17.3, 1.5 Hz), 5.08 (2H, dd, J = 10.8, 1.5 Hz),
152.2, 151.0, 146.6, 144.7, 142.6, (300 MHz, CDCl3)
δ 7.05 (2H, dd, J = 17.3, 10.8 Hz), 6.78 (2H, s),
109.7 (CH), 61.3, 61.1, 56.3, 56.0 (CH3).
3.87 (6H, s), 3.81 (6H, s), 3.50 (6H, s), 0.60 (6H, s); 13C NMR (75
MHz, CDCl3) 158.0 (x 2), 154.3 (x 2), 141.1 (x 2), 139.9 (x 2),
δ
2-Bromo-3,4,5-trimethoxy-1-vinylbenzene (9). A solution of
124.6 (x 2) (C), 139.2 (x 2), 106.0 (x 2) (CH), 114.2 (x 2) (CH2),
60.6 (x 2), 60.1 (x 2), 55.9 (x 2), 5.0 (x 2) (CH3); HR ESMS m/z
467.1873 (M+Na+). Calcd. for C24H32NaO6Si, 467.1866.
methyl triphenylphosphonium iodide (1 mmol, 404 mg) in dry
THF (4 mL) was treated under an inert atmosphere with 2.5M
n-BuLi (1 mmol, 0.4 mL). The mixture was then stirred for 20
min. at room temperature. A solution of 2-bromo-3,4,5- Dimethyl bis(2,3,4-trimethoxy-6-vinylphenyl)germane
(14b).
trimethoxy-benzaldehyde (1 mmol) in dry THF (3 mL) is then Compound 9 and Me2GeCl2 (1 mmol) were allowed to react
added dropwise, and the mixture is stirred at room under the same reaction conditions as above for 14a. Work-up
temperature for 2 h. After this time, satd aq NH4Cl (5 mL) was gave an oily residue which was chromatographed on silica gel
added and the reaction mixture was extracted with CH2Cl2. The (elution with hexane-Et2O 95:5) to yield 14b (43%): off-white
1
organic layer was washed three times with brine and finally gum; H NMR (300 MHz, CDCl3)
δ 6.86 (2H, dd, J = 17.3, 10.8
dried over anhydrous MgSO4. Removal of volatiles under Hz), 6.73 (2H, s), 5.33 (2H, dd, J = 17.3, 1.5 Hz), 5.00 (2H, dd, J =
reduced pressure gave an oily residue which was 10.8, 1.5 Hz), 3.80 (6H, s), 3.75 (6H, s), 3.52 (6H, s), 0.67 (6H, s);
chromatographed on silica gel (elution with hexane-EtOAc 9:1) 13C NMR (75 MHz, CDCl3)
δ
157.0 (x 2), 154.0 (x 2), 141.2 (x 2),
7.04 (1H, dd, J 139.2 (x 2), 127.1 (x 2) (C), 138.6 (x 2), 105.6 (x 2) (CH), 114.1 (x
= 17.3, 10.8 Hz), 6.89 (1H, s), 5.60 (1H, dd, J = 17.3, 1 Hz), 5.31 2) (CH2), 60.7 (x 2), 60.5 (x 2), 55.9 (x 2), 5.1 (x 2) (CH3); HR
(1H, dd, J = 10.8, 1 Hz), 3.88 (9H, s); 13C NMR (75 MHz, CDCl3) ESMS m/z 513.1315 (M+Na+). Calcd. for C24H3274GeNaO6,
to yield
9 δ
(74%): oil; 1H NMR (300 MHz, CDCl3)
δ
152.8, 150.9, 143.1, 133.3, 110.6 (C), 136.0, 105.3 (CH), 116.0 513.1308.
(CH2), 61.3, 61.0, 56.2 (CH3).
Dimethyl bis(2,3,4-trimethoxy-6-vinylphenyl)stannane
Compound and Me2SnCl2 (1 mmol) were allowed to react
(2.16 mmol, 500 mg) was dissolved in dry under the same reaction conditions as above for 14a. Work-up
CH2Cl2 (12 mL), cooled to 0°C and treated with triethylamine gave an oily residue which was chromatographed on silica gel
(5.40 mmol, 752 L) and triphenylmethyl chloride (4.30 mmol, (elution with hexane-Et2O 95:5) to yield 14c (57%): off-white
1.2 g). The mixture was then stirred for 14 h at room gum; 1H NMR (300 MHz, CDCl3)
6.90 (2H, s), 6.84 (2H, dd, J =
(14c).
2-Bromo-3,4-dimethoxy-5-(triphenylmethyl)oxy-1-vinylbenzene
10). Aldehyde
9
(
3
ꢀ
δ
temperature. After this time, satd aq NH4Cl (5 mL) was added 17.3, 10.8 Hz), 5.48 (2H, dd, J = 17.3, 1.5 Hz), 5.10 (2H, dd, J =
and the reaction mixture was extracted with CH2Cl2. The 10.8, 1.5 Hz), 3.88 (6H, s), 3.82 (6H, s), 3.65 (6H, s), 0.55 (6H, s);
organic layer was washed three times with brine and finally 13C NMR (75 MHz, CDCl3)
δ 157.2 (x 2), 154.4 (x 2), 140.7 (x 2),
dried over anhydrous MgSO4. Removal of volatiles under 140.5 (x 2), 128.8 (x 2) (C), 139.4 (x 2), 105.0 (x 2) (CH), 114.0 (x
reduced pressure gave an oily residue of the tritylated 2) (CH2), 60.7 (x 2), 60.6 (x 2), 56.0 (x 2), −2.6 (x 2) (CH3); HR
aldehyde, which was then subjected to Wittig methylenation ESMS m/z 559.1124 (M+Na+). Calcd. for C24H32NaO6120Sn,
under the conditions described above to yield 10 (60%): 559.1118.
1
yellowish gum; H NMR (300 MHz, CDCl3)
δ 7.53-7.48 (6H, m),
2,3,4,6,7,8-Hexamethoxy-5,5-dimethyl-5H-dibenzo[b,f]silepine
15a). The second-generation Hoveyda-Grubbs ruthenium
7.35-7.25 (9H, m), 6.88 (1H, s), 6.85 (1H, s), 6.78 (1H, dd, J =
17.3, 10.8 Hz), 5.03 (1H, dd, J = 10.8, 1 Hz), 4.98 (1H, dd, J =
(
catalyst (0.01 mmol, 6.3 mg) was disolved under Ar in dry,
deoxygenated toluene (40 mL). A solution of diolefin 14a (0.1
mmol) in dry, deoxygenated toluene (27 mL) was then added
dropwise. The mixture was then heated at reflux until
17.3, 1 Hz), 3.62 (3H, s); 13C NMR (75 MHz, CDCl3)
δ 152.8,
145.2, 144.0 (x 3), 128.7, 116.0, 91.6 (C), 135.2, 129.3 (x 6),
This journal is © The Royal Society of Chemistry 20
J. Name., 2013, 00, 1-3 | 9
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 10 of 13
View Article Online
DOI: 10.1039/C8OB01148F
ARTICLE
Journal Name
consumption of the starting material (TLC monitoring). After 17.3, 10.8 Hz), 5.47 (2H, dd, J = 17.3, 1.2 Hz), 5.04 (2H, dd, J =
the required time, the volatiles were removed under reduced 10.8, 1.2 Hz), 3.92 (6H, s), 3.83 (6H, s), 0.60 (6H, s); 13C NMR
pressure and the oily residue was chromatographed on silica (75 MHz, CDCl3) δ 150.2 (x 2), 148.2 (x 2), 137.5 (x 2), 128.7 (x
gel (elution with hexane-Et2O 95:5) to yield 15a (86%): off- 2) (C), 137.7 (x 2), 117.5 (x 2), 108.4 (x 2) (CH), 113.1 (x 2)
1
white solid, mp 200-202°C; H NMR (300 MHz, CDCl3)
(2H, s), 6.67 (2H, s), 3.87 (6H, s), 3.85 (6H, s), 3.84 (6H, s), 0.58
(6H, s); 13C NMR (75 MHz, CDCl3)
157.6 (x 2), 153.8 (x 2),
δ
6.84 (CH2), 56.0 (x 2), 55.8 (x 2), 0.4 (x 2) (CH3).
Bis(4,5-dimethoxy-2-vinylphenyl)dimethylgermane
(22b).
δ
Prepared in 34% yield from 19 under the same reaction
1
conditions as in the synthesis of 14b: off-white gum; H NMR
142.3 (x 2), 137.6 (x 2), 123.5 (x 2) (C), 133.0 (x 2), 109.4 (x 2)
(CH), 61.4 (x 2), 60.8 (x 2), 56.0 (x 2), 1.0 (x 2) (CH3); HR ESMS
m/z 439.1554 (M+Na+). Calcd. for C22H28NaO6Si, 439.1553.
(300 MHz, CDCl3) δ 7.15 (2H, s), 6.88 (2H, s), 6.77 (2H, dd, J =
17.3, 10.8 Hz), 5.52 (2H, dd, J = 17.3, 1.2 Hz), 5.08 (2H, dd, J =
10.8, 1.2 Hz), 3.92 (6H, s), 3.80 (6H, s), 0.72 (6H, s); 13C NMR
2,3,4,6,7,8-Hexamethoxy-5,5-dimethyl-5H-
dibenzo[b,f]germepine (15b). Prepared from diolefin 14b under (75 MHz, CDCl3) δ 149.9 (x 2), 148.5 (x 2), 136.4 (x 2), 131.0 (x
the same reaction conditions as for 15a. Chromatography on 2) (C), 137.6 (x 2), 116.7 (x 2), 108.2 (x 2) (CH), 113.0 (x 2)
silica gel (elution with hexane-Et2O 95:5) gave 15b (90%): off- (CH2), 55.9 (x 2), 55.8 (x 2), 0.3 (x 2) (CH3).
1
white solid, mp 202-204°C; H NMR (300 MHz, CDCl3)
δ 6.76
Bis(4,5-dimethoxy-2-vinylphenyl)dimethylstannane
(22c).
(2H, s), 6.65 (2H, s), 3.86 (6H, s), 3.85 (6H, s), 3.84 (6H, s), 0.74
(6H, s); 13C NMR (75 MHz, CDCl3)
156.7 (x 2), 153.6 (x 2),
Prepared in 50% yield from 19 under the same reaction
1
conditions as in the synthesis of 14c: off-white gum; H NMR
δ
142.1 (x 2), 137.1 (x 2), 126.0 (x 2) (C), 133.1 (x 2), 109.4 (x 2)
(CH), 61.3 (x 2), 60.8 (x 2), 56.0 (x 2), 1.3 (x 2) (CH3); HR ESMS
m/z 485.0976 (M+Na+). Calcd. for C22H2874GeNaO6, 485.0995.
(300 MHz, CDCl3) δ 7.18 (2H, s), 6.88 (2H, s), 6.71 (2H, dd, J =
17.3, 10.8 Hz), 5.55 (2H, dd, J = 17.3, 1.2 Hz), 5.14 (2H, dd, J =
10.8, 1.2 Hz), 3.92 (6H, s), 3.79 (6H, s), 0.57 (6H, s); 13C NMR
2,3,4,6,7,8-Hexamethoxy-5,5-dimethyl-5H-dibenzo[b,f]stannepine (75 MHz, CDCl3) δ 150.0 (x 2), 148.6 (x 2), 137.9 (x 2), 132.5 (x
15c). Prepared from diolefin 14c under the same reaction 2) (C), 139.2 (x 2), 118.7 (x 2), 106.0 (x 2) (CH), 113.3 (x 2)
(
conditions as for 15a. Chromatography on silica gel (elution (CH2), 55.9 (x 2), 55.8 (x 2), −6.9 (x 2) (CH3).
with hexane-Et2O 95:5) gave 15c (21%): off-white solid, mp
Dimethyl bis(6-vinylbenzo[d][1,3]dioxol-5-yl)silane (23a). Prepared
173-175 °C; 1H NMR (300 MHz, CDCl3)
δ
6.66 (2H, s), 6.65 (2H,
s), 3.86 (6H, s), 3.85 (6H, s), 3.84 (6H, s), 0.57 (6H, s); 13C NMR
(75 MHz, CDCl3) 156.9 (x 2), 154.0 (x 2), 141.2 (x 2), 139.0 (x
in 36% yield from 20 under the same reaction conditions as in
the synthesis of 14a: off-white gum; 1H NMR (300 MHz, CDCl3)
δ
δ
7.07 (2H, s), 6.97 (2H, s), 6.77 (2H, dd, J = 17.3, 10.8 Hz), 5.96
(4H, s), 5.43 (2H, dd, J = 17.3, 1.2 Hz), 5.02 (2H, dd, J = 10.8, 1.2
Hz), 0.55 (6H, s); 13C NMR (75 MHz, CDCl3)
149.2 (x 2), 147.2
(x 2), 138.9 (x 2), 130.4 (x 2) (C), 137.3 (x 2), 113.9 (x 2), 106.0
19). Prepared in 85% (x 2) (CH), 113.5, 101.0 (x 2) (CH2), 0.3 (x 2) (CH3).
yield by means of Wittig methylenation of aldehyde 16 as
2), 127.7 (x 2) (C), 133.4 (x 2), 110.3 (x 2) (CH), 61.1 (x 2), 60.8
(x 2), 56.1 (x 2), −6.4 (x 2) (CH3); HR ESMS m/z 531.0818
(M+Na+). Calcd. for C22H28NaO6120Sn, 531.0805.
δ
1-Bromo-4,5-dimethoxy-2-vinylbenzene
(
Dimethyl bis(6-vinylbenzo[d][1,3]dioxol-5-yl)germane (23b).
1
described for the synthesis of
7.03 (1H, s), 6.99 (1H, s), 6.97 (1H, dd, J = 17.3, 10.8 Hz), 5.58
(1H, dd, J = 17.3, 1 Hz), 5.26 (1H, dd, J = 10.8, 1 Hz), 3.89 (3H,
s), 3.86 (3H, s); 13C NMR (75 MHz, CDCl3)
149.5, 148.7, 129.6,
9: oil; H NMR (300 MHz, CDCl3)
Prepared in 41% yield from 20 under the same reaction
1
conditions as in the synthesis of 14b: off-white gum; H NMR
δ
(300 MHz, CDCl3)
δ 7.12 (2H, s), 6.88 (2H, s), 6.73 (2H, dd, J =
δ
17.3, 10.8 Hz), 5.96 (4H, s), 5.48 (2H, dd, J = 17.3, 1.2 Hz), 5.07
(2H, dd, J = 10.8, 1.2 Hz), 0.68 (6H, s); 13C NMR (75 MHz, CDCl3)
114.4 (C), 135.5, 115.4, 108.8 (CH), 114.7 (CH2), 56.3, 56.1
(CH3).
δ 149.0 (x 2), 147.3 (x 2), 137.7 (x 2), 132.6 (x 2) (C), 137.3 (x 2),
(20). Prepared in 79% 113.3 (x 2), 105.8 (x 2) (CH), 113.5, 101.0 (x 2) (CH2), 0.2 (x 2)
5-Bromo-6-vinylbenzo[d][1,3]dioxole
yield by means of Wittig methylenation of aldehyde 17 as (CH3).
1
described for the synthesis of
7.03 (1H, s), 6.99 (1H, s), 6.98 (1H, dd, J = 17.3, 10.8 Hz), 5.97
(2H, s), 5.55 (1H, dd, J = 17.3, 1 Hz), 5.26 (1H, dd, J = 10.8, 1
Hz); 13C NMR (75 MHz, CDCl3)
148.2, 147.8, 131.0, 114.8 (C),
135.6, 112.7, 106.1 (CH), 115.1, 101.9 (CH2).
9: oil; H NMR (300 MHz, CDCl3)
Dimethyl bis(6-vinylbenzo[d][1,3]dioxol-5-yl)stannane (23c).
δ
Prepared in 47% yield from 20 under the same reaction
1
conditions as in the synthesis of 14c: off-white gum; H NMR
δ
(300 MHz, CDCl3)
17.3, 10.8 Hz), 5.95 (4H, s), 5.53 (2H, dd, J = 17.3, 1 Hz), 5.13
this (2H, dd, J = 10.8, 1 Hz), 0.55 (6H, s); 13C NMR (75 MHz, CDCl3)
δ 7.15 (2H, s), 6.87 (2H, s), 6.67 (2H, dd, J =
1-Bromo-4-fluoro-2-vinylbenzene (21).
Although
δ
compound is commercial, we prepared it as an oil in 47% yield 149.2 (x 2), 147.4 (x 2), 139.1 (x 2), 134.1 (x 2) (C), 138.8 (x 2),
by means of Wittig methylenation of aldehyde 18 as described 113.8 (x 2), 105.8 (x 2) (CH), 115.3, 101.0 (x 2) (CH2), −6.8 (x 2)
for the synthesis of 9. Its spectral data are in good agreement (CH3).
with those described in the literature.28
Bis(4-fluoro-2-vinylphenyl)dimethylsilane (24a). Prepared in
Bis(4,5-dimethoxy-2-vinylphenyl)dimethylsilane
(22a). 20% yield from 21 under the same reaction conditions as in
Prepared in 36% yield from 19 under the same reaction the synthesis of 14a: oil; 1H NMR (300 MHz, CDCl3)
δ 7.47 (2H,
conditions as in the synthesis of 14a: off-white gum; H NMR dd, J = 8.3, 6.5 Hz), 7.22 (2H, dd, J = 10.7, 2.5 Hz), 6.97 (2H, td, J
1
(300 MHz, CDCl3)
δ 7.11 (2H, s), 6.98 (2H, s), 6.80 (2H, dd, J = = 8.3, 2.5 Hz), 6.75 (2H, ddd, J = 17.3, 10.8, 1.7 Hz), 5.55 (2H,
10 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 11 of 13
View Article Online
DOI: 10.1039/C8OB01148F
Journal Name
dd, J = 17.3, 1 Hz), 5.13 (2H, dd, J = 10.8, 1 Hz), 0.58 (6H, s); 13
ARTICLE
1
oil; H NMR (300 MHz, CDCl3)
C
δ
6.98 (2H, s), 6.85 (2H, s), 6.78
164.6 (x 2, center point of doublet, (2H, s), 5.93 (4H, s), 0.43 (6H, s); 13C NMR (75 MHz, CDCl3)
δ δ
NMR (75 MHz, CDCl3)
1JC−F = 246.5 Hz), 146.6 (x 2, doublet, JC−F = 7 Hz), 132.2 (x 2, 148.6 (x 2), 147.8 (x 2), 136.4 (x 2), 130.5 (x 2) (C), 131.7 (x 2),
3
4
3
doublet, JC−F = 3 Hz) (C), 137.0 (x 2, doublet, JC−F = 7.5 Hz), 111.4 (x 2), 110.0 (x 2) (CH), 101.0 (CH2), −4.2 (x 2) (CH3); HR
136.9 (x 2, doublet, 4JC−F = 2 Hz), 114.2 (x 2, doublet, 2JC−F = 19.5 ESMS m/z 325.0886 (M+H+). Calcd. for C18H17O4Si, 325.0896.
2
Hz), 112.4 (x 2, doublet, JC−F = 20.5 Hz) (CH), 116.3 (x 2) (CH2),
Germepine 26b. Prepared from diolefin 23b under the same
0.0 (x 2) (CH3); 19F NMR (282 MHz, CDCl3)
δ
−112.4.
reaction conditions as in the synthesis of 15a. Chromatography
Bis(4-fluoro-2-vinylphenyl)dimethylgermane
(
24b). Prepared on silica gel (elution with hexane-Et2O 95:5) gave 26b (80%):
1
in 22% yield from 21 under the same reaction conditions as in oil; H NMR (300 MHz, CDCl3)
δ 6.90 (2H, s), 6.83 (2H, s), 6.71
1
the synthesis of 14b: oil; H NMR (300 MHz, CDCl3)
δ δ
7.37 (2H, (2H, s), 5.92 (4H, s), 0.56 (6H, s); 13C NMR (75 MHz, CDCl3)
dd, J = 8.3, 6.5 Hz), 7.27 (2H, dd, J = 10.7, 2.5 Hz), 6.95 (2H, td, J 148.3 (x 2), 147.8 (x 2), 135.6 (x 2), 132.8 (x 2) (C), 131.9 (x 2),
= 8.3, 2.5 Hz), 6.73 (2H, ddd, J = 17.2, 10.8, 1.7 Hz), 5.59 (2H, 111.0 (x 2), 109.8 (x 2) (CH), 101.0 (CH2), −4.8 (x 2) (CH3); HR
dd, J = 17.2, 1 Hz), 5.18 (2H, dd, J = 10.8, 1 Hz), 0.70 (6H, s); 13C ESMS m/z 371.0358 (M+H+). Calcd. for C18H1774GeO4, 371.0340.
NMR (75 MHz, CDCl3)
δ 164.1 (x 2, center point of doublet,
Stannepine 26c. Prepared from diolefin 23c under the same
reaction conditions as in the synthesis of 15a. Chromatography
3
1JC−F = 246 Hz), 145.7 (x 2, doublet, JC−F = 6.7 Hz), 134.3 (x 2,
4
4
doublet, JC−F = 3.3 Hz) (C), 136.8 (x 2, doublet, JC−F = 2.7 Hz),
136.0 (x 2, doublet, 3JC−F = 7.7 Hz), 114.4 (x 2, doublet, 2JC−F = 20
on silica gel (elution with hexane-Et2O 95:5) gave 26c (23%):
1
oil; H NMR (300 MHz, CDCl3)
δ 6.90 (2H, s), 6.83 (2H, s), 6.71
2
Hz), 112.3 (x 2, doublet, JC−F = 20.5 Hz) (CH), 116.4 (x 2) (CH2),
(2H, s), 5.92 (4H, s), 0.56 (6H, s); 13C NMR (75 MHz, CDCl3)
δ
148.4 (x 2), 147.5 (x 2), 137.5 (x 2), 134.3 (x 2) (C), 133.1 (x 2),
24c). Prepared 113.2 (x 2), 109.7 (x 2) (CH), 100.9 (CH2), −11.2 (x 2) (CH3); HR
−0.2 (x 2) (CH3); 19F NMR (282 MHz, CDCl3)
δ −113.4.
Bis(4-fluoro-2-vinylphenyl)dimethylstannane
(
in 25% yield from 21 under the same reaction conditions as in ESMS m/z 417.0110 (M+H+). Calcd. for C18H17O4120Sn,
1
the synthesis of 14c: oil; H NMR (300 MHz, CDCl3)
δ 7.35 (2H, 417.0149.
dd, J = 8, 6.5 Hz), 7.30 (2H, dd, J = 10.7, 2.5 Hz), 6.95 (2H, td, J =
2,8-Difluoro-5,5-dimethyl-5H-dibenzo[b,f]silepine
(27a).
8, 2.5 Hz), 6.70 (2H, ddd, J = 17.2, 10.8, 1.7 Hz), 5.63 (2H, dd, J
= 17.2, 1 Hz), 5.26 (2H, dd, J = 10.8, 1 Hz), 0.57 (6H, s); 13C NMR
Prepared from diolefin 24a under the same reaction conditions
as in the synthesis of 15a. Chromatography on silica gel
(elution with hexane-Et2O 95:5) gave 27a (80%): off-white
solid, mp 102-104°C; 1H NMR (300 MHz, CDCl3)
(2H, m), 7.10-7.00 (4H, m), 6.92 (2H, s), 0.48 (6H, s); 13C NMR
1
(75 MHz, CDCl3)
δ
164.2 (x 2, center point of doublet, JC−F
=
245 Hz), 147.2 (x 2, doublet, 3JC−F = 6.7 Hz), 135.8 (x 2, doublet,
4JC−F = 3.5 Hz) (C), 138.3 (x 2, doublet, 4JC−F = 2.6 Hz), 138.2 (x 2,
doublet, 3JC−F = 7 Hz), 114.6 (x 2, doublet, 2JC−F = 19.5 Hz), 112.2
δ 7.55-7.50
1
163.7 (x 2, center point of doublet, JC−F
(75 MHz, CDCl3)
δ
246 Hz), 143.4 (x 2, doublet, JC−F = 7 Hz), 133.2 (x 2, doublet,
=
2
(x 2, doublet, JC−F = 20.5 Hz) (CH), 116.7 (x 2) (CH2), −7.2 (x 2)
(CH3); 19F NMR (282 MHz, CDCl3)
3
δ
−113.4.
3
4JC−F = 3 Hz) (C), 134.6 (x 2, doublet, JC−F = 7.5 Hz), 133.1 (x 2,
2,3,7,8-Tetramethoxy-5,5-dimethyl-5H-dibenzo[b,f]silepine
doublet, 4JC−F = 2 Hz), 116.2 (x 2, doublet, 2JC−F = 20 Hz), 114.9 (x
2
(
25a). Prepared from diolefin 22a under the same reaction 2, doublet, JC−F = 20 Hz) (CH), −4.2 (x 2) (CH3); 19F NMR (282
conditions as in the synthesis of 15a. Chromatography on silica MHz, CDCl3)
gel (elution with hexane-Et2O 95:5) gave 25a (90%): off-white
δ −113.3.
2,8-Difluoro-5,5-dimethyl-5H-dibenzo[b,f]germepine
(27b).
solid, mp 200-202 ºC; 1H NMR (300 MHz, CDCl3)
δ
7.01 (2H, s),
6.89 (2H, s), 6.85 (2H, s), 3.92 (6H, s), 3.88 (6H, s), 0.48 (6H, s);
13C NMR (75 MHz, CDCl3)
149.6 (x 2), 149.0 (x 2), 135.3 (x 2),
Prepared from diolefin 24b under the same reaction conditions
as in the synthesis of 15a. Chromatography on silica gel
(elution with hexane-Et2O 95:5) gave 27b (70%): off-white
δ
129.0 (x 2) (C), 131.7 (x 2), 114.6 (x 2), 112.8 (x 2) (CH), 56.1 (x
2), 55.9 (x 2), −4.1 (x 2) (CH3); HR ESMS m/z 379.1347 (M+Na+).
Calcd. for C20H24NaO4Si, 379.1342.
solid, mp 98-100°C; 1H NMR (300 MHz, CDCl3)
δ 7.45-7.40 (2H,
m), 7.10-7.00 (4H, m), 6.86 (2H, s), 0.60 (6H, s); 13C NMR (75
1
163.4 (x 2, center point of doublet, JC−F = 245
MHz, CDCl3)
δ
2,3,7,8-Tetramethoxy-5,5-dimethyl-5H-
Hz), 143.0 (x 2, doublet, 3JC−F = 6.7 Hz), 135.3 (x 2, doublet, 4JC−F
3
25b). Prepared from diolefin 22b = 3.5 Hz) (C), 133.8 (x 2, doublet, JC−F = 7.5 Hz), 133.2 (x 2,
dibenzo[b,f]germepine
(
under the same reaction conditions as in the synthesis of 15a
Chromatography on silica gel (elution with hexane-Et2O 95:5) (x 2, doublet, JC−F = 19.5 Hz) (CH), −4.8 (x 2) (CH3); 19F NMR
.
doublet, 4JC−F = 2.5 Hz), 116.2 (x 2, doublet, 2JC−F = 20 Hz), 114.9
2
1
gave 25b (78%): off-white solid, mp 203-205 ºC; H NMR (300 (282 MHz, CDCl3)
δ
−114.2.
MHz, CDCl3)
s), 3.87 (6H, s), 0.60 (6H, s); 13C NMR (75 MHz, CDCl3)
δ
6.92 (2H, s), 6.88 (2H, s), 6.78 (2H, s), 3.91 (6H,
149.2
2,8-Difluoro-5,5-dimethyl-5H-dibenzo[b,f]stannepine
(
27c).
δ
Prepared from diolefin 24c under the same reaction conditions
as in the synthesis of 15a. Chromatography on silica gel
(elution with hexane-Et2O 95:5) gave 27c (28%): off-white
solid, mp 96-98°C; 1H NMR (300 MHz, CDCl3)
δ 7.40 (2H, dd, J =
(x 2), 149.0 (x 2), 134.7 (x 2), 131.3 (x 2) (C), 132.0 (x 2), 114.2
(x 2), 112.7 (x 2) (CH), 56.1 (x 2), 56.0 (x 2), −4.7 (x 2) (CH3); HR
ESMS m/z 403.0969 (M+H+). Calcd. for C20H2574GeO4, 403.0965.
Silepine 26a. Prepared from diolefin 23a under the same 8, 6.5 Hz), 7.03 (2H, dd, J = 10.8, 2.5 Hz), 6.98 (2H, td, J = 8.5,
reaction conditions as in the synthesis of 15a. Chromatography 2.5 Hz), 6.86 (2H, s), 0.45 (6H, s); 13C NMR (75 MHz, CDCl3)
on silica gel (elution with hexane-Et2O 95:5) gave 26a (86%):
δ
163.5 (x 2, center point of doublet, 1JC−F = 245 Hz), 145.1 (x 2,
This journal is © The Royal Society of Chemistry 20
J. Name., 2013, 00, 1-3 | 11
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 12 of 13
View Article Online
DOI: 10.1039/C8OB01148F
ARTICLE
Journal Name
3
doublet, JC−F = 6.5 Hz), 136.9 (x 2, doublet, JC−F = 3.5 Hz) (C),
4
tubulin antibody (dilution 1:400 in PBS-BSA 1% from a 1 mg/mL
solution; monoclonal antibody, clone DM1A, Sigma-Aldrich). Next,
cells were washed with PBS and incubated for 30 min at room
temperature in darkness with Hoechst 2 mM in water. Then, cells
were washed in PBS and coverglasses were mounted with 10 μL of
Glycine/Glycerol buffer. The cytoskeleton was imaged by a confocal
laser scanning microscope (CLSM) Leica SP5 with a Leica inverted
microscope, equipped with a Plan-Apochromat 63 oil immersion
objective (NA=1.4). Each image was recorded with the CLSM’s
spectral mode selecting specific domains of the emission spectrum.
The FITC fluorophore was excited at 488 nm with an argon laser and
its fluorescence emission was collected between 496 nm and 535
nm.
3
136.3 (x 2, doublet, JC−F = 7.5 Hz), 134.0 (x 2, doublet, JC−F
4
=
2
2.7 Hz), 116.0 (x 2, doublet, JC−F = 20 Hz), 114.7 (x 2, doublet,
2JC−F = 19 Hz) (CH), −11.2 (x 2) (CH3); 19F NMR (282 MHz, CDCl3)
δ
−114.1.
Biological studies. Materials and methods
Cell culture
Cell culture media were purchased from Gibco (Grand Island,
NY). Fetal bovine serum (FBS) was a product of Harlan-Seralab
(Belton, U.K.). Supplements and other chemicals not listed in this
section were obtained from Sigma Chemical Co. (St. Louis, MO).
Plastics for cell culture were supplied by Thermo Scientific BioLite.
All tested compounds were dissolved in DMSO at a concentration of
20 μM and stored at -20°C until use.
Tube formation inhibition assay
Wells of a 96-well μ-plate for angiogenesis were coated with 12
μL of Matrigel (10 mg/mL, BD Biosciences) at 4ºC. After
gelatinization at 37ºC for 30 min, HMEC-1 cells were seeded at 2 x
104 cells/well in 25 μL of culture medium on top of the Matrigel and
were incubated 30 min at 37ºC while are attached. Then,
compounds were added dissolved in 25 μL of culture medium and
after 20 h of incubation at 37ºC, tube destruction was evaluated
and photographed.
HT-29, MCF-7, HeLa and HEK-293 cell lines were maintained in
Dulbecco’s modified Eagle’s medium (DMEM) containing glucose
(1g/L), glutamine (2 mM), penicillin (50 IU/mL), streptomycin (50
μg/mL), and amphotericin B (1.25 μg/mL), supplemented with 10%
FBS. HMEC-1 cell line was maintained in Dulbecco’s modified
Eagle’s medium (DMEM) low glucose, glutamine (2 mM), penicillin
(50 IU/mL), streptomycin (50 μg/mL), and amphotericin B (1.25
μg/mL), supplemented with 10% FBS.
Phospho-VEGFR2 quantification by ELISA
MTT assay
A total of 5 × 103 HT-29, MCF-7, HeLa or HEK-293 cells in a total
volume of 100 μL of their respective growth media were incubated
with serial dilutions of the tested compounds. The 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma
Chemical Co.) dye reduction assay in 96-well microplates was used,
as previously described [26]. After 2 days of incubation (37 °C, 5%
CO2 in a humid atmosphere), 10 μL of MTT (5 mg/mL in phosphate-
buffered saline, PBS) was added to each well, and the plate was
incubated for a further 3 h (37 °C). The supernatant was discarded
and replaced by 100 µL of DMSO to dissolve formazan crystals. The
absorbance was then read at 490 nm by spectrophotometry. For all
concentrations of compound, cell viability was expressed as the
percentage of the ratio between the mean absorbance of treated
cells and the mean absorbance of untreated cells. Three
independent experiments were performed, and the IC50 values (i.e.,
concentration half inhibiting cell proliferation) were graphically
determined.
HMEC-1 cells were seeded at 5·105 cells/well in 6-well plates
and once they were at 80% of their confluency, they were starved
with medium containing 0.1% of FBS for 24 h. Then, cells were
incubated with the corresponding compounds for 2 hours and next
cells were stimulated with 50 ng/ml of Recombinant VEGF-165 for
30 minutes at 37ºC. After that, lysates were collected, protein
quantification was carried out by Bradford test and, then, and
phospho-VEGFR2 was quantified using PathScan® Phospho-
VEGFR2(Tyr1175) Sandwich ELISA Kit according to the
manufacturer’s instructions.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Cell cycle analysis
Financial support has been granted by the Spanish Government
Progression of the cell cycle was analysed by means of flow
cytometry with propidium iodide. After incubation with compounds
for 24 h, A549 cells were fixed, treated with RNase and stained with
propidium iodide following instructions of BD CycletestTM DNA Kit.
Analysis was performed with a BD AccuriTM C6 flow cytometer.
(MINECO, Ministerio de Economía
y Competitividad, project
CTQ2014-52949-P) and by the Universitat Jaume I (project PI-1B-
2015-75). V. B. thanks the Conselleria d’Educaciò, Investigaciò,
Cultura i Esport de la Generalitat Valenciana for financial support.
Microtubule network study by immunofluorescence
Notes and references
1
(a) D. A. Dias, S. Urban and U. Roessner, Metabolites, 2012,
, 303-336. (b) T. Rodrigues, Org. Biomol. Chem., 2017, 15
9275-9282.
M. Aeluri, S. Chamakuri, B. Dasari, S. K. R. Guduru, R.
Jimmidi, S. Jogula and P. Arya, Chem. Rev., 2014, 114, 4640-
4694.
Immunofluorescent analysis of the microtubule network was
performed on the A-549 cell line. In this assay, 1,5x105 cells were
2
,
plated on
a coverglass and incubated with the different
2
concentrations of selected compounds for 16 h. Cells were then
washed with PEMP, permeabilized with PEM-Triton X-100 0.5% for
90 seconds at room temperature and fixed in 3.7% formaldehyde (in
PEM pH 7.4) for 30 min at rt. Direct immunostaining was carried out
for 2.5 h at 37°C in darkness with primary FITC-conjugated anti- -
3
4
J. Hong, Chem. Eur. J., 2014, 20, 10204-10212.
(a) H. Yuan, Q. Ma, L. Ye and G. Piao, Molecules, 2016, 21
559.
,
12 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20
Please do not adjust margins

Please do not adjust margins
Organic & Biomolecular Chemistry
Page 13 of 13
View Article Online
DOI: 10.1039/C8OB01148F
Journal Name
ARTICLE
5
(a) S. Medici, M. Peana, V. M. Nurchi, J. I. Lachowicz, G. 20 T. Matsuda and S. Sato, J. Org. Chem., 2013, 78, 3329-3335.
Crisponi and M. A. Zoroddu, Coord. Chem. Rev., 2015, 284,
329-350. (b) T. C. Johnstone, K. Suntharalingam and S. J.
21 A. H. Hoveyda, D. G. Gillingham, J. J. Van Veldhuizen, O.
Kataoka, S. B. Garber, J. S. Kingsbury and J. P. A. Harrity, Org.
Lippard, Chem. Rev., 2016, 116, 3436-3486.
For a recent interesting review on natural product 22 F. Alonso, P. Riente and M. Yus, Eur. J. Org. Chem., 2009,
Biomol. Chem., 2004, 2, 8-23.
6
7
analogues: M. E. Maier, Org. Biomol. Chem., 2015, 13, 5302-
5343.
6034-6042.
23 V. Chaudhary, J. B. Venghateri, H. P. S. Dhaked, A. S. Bhoyar,
S. K. Guchhait and D. Panda, J. Med. Chem. 2016, 59, 3439-
3451.
(a) A. Marzo-Mas, P. Barbier, G. Breuzard, D. Allegro, E,
Falomir, J. Murga, M. Carda, V. Peyrot and J. A. Marco, Eur. J.
Med. Chem., 2017, 126, 526-535; (b) V. Blasco, A. C. Cuñat, J. 24 M. Su, J. Huang, S. Liu, Y. Xiao, X. Qin, J. Liu, C. Pi, T. Luo, J. Li,
F. Sanz-Cervera, J. A. Marco, E. Falomir, J. Murga and M.
Carda, Eur. J. Med. Chem., 2018, 150, 817-828.
For a review: T. Shen, X.-N. Wang and H.-X. Lou, Nat. Prod.
Rep., 2009, 26, 916-935.
X. Chen and Z. Luo, Sci. Rep. 2016, 6, 28139.
25 Sorafenib was prepared following a published procedure: D.
Bankston, J. Dumas, R. Natero, B. Riedl, M.-K. Monahan and
R. Sibley, Org. Proc. Res. Dev., 2002, 6, 777-781.
8
9
Y. Shan, J. Zhang, Z. Liu, M. Wang and Y. Dong, Curr. Med.
Chem., 2011, 18, 523-538
10 (a) M. Marrelli, F. Conforti, G. A. Statti, X. Cachet, S. Michel,
26 S. Wilhelm, C. Carter, M. Lynch, T. Lowinger, J. Dumas, R.A.
Smith, B. Schwartz, R. Simantov and S. Kelley, Nat. Rev. Drug
Discov. 2006, 5, 835-844.
F. Tillequin and F. Menichini, Curr. Med. Chem., 2011, 18
3035-3081. (b) C. B. Pattillo, Drugs Fut., 2011, 36, 385-390.
,
27 For toxicity issues related to tin-containing drugs, see: (a) T.
S. B. Baul and D. de Vos, Invest. New Drugs, 2010, 28, 609-
614; (b) A. Varela-Ramirez, M. Costanzo, Y. P. Carrasco, K. H.
Pannell, R. J. Aguilera, Cell Biol. Toxicol., 2011, 27, 159-
168;(c) J. B. Graceli, G. C. Sena, P. F. I. Lopes, G. C.
Zamprogno, M. B. da Costa, A. F. L. Godoi, D. M. dos Santos,
M. R. Rodrigues de Marchi and M. A. dos Santos Fernandez,
Reprod. Toxicol., 2013, 36, 40-52.
11 P. O. Patil, A. G. Patil, R. A. Rane, P. C. Patil, P. K. Deshmukh,
S. B. Bari, D. A. Patil and S. S. Naphade, Anti-Cancer Agents in
Medicinal Chemistry, 2015, 15, 955-969.
12 (a) S. Torijano-Gutiérrez, S. Díaz-Oltra, E. Falomir, J. Murga,
M. Carda and J.A. Marco, Bioorg. Med. Chem., 2013, 21,
7267-7274; (b) S. Torijano-Gutiérrez, C. Vilanova, S. Díaz-
Oltra, J. Murga, E. Falomir, M. Carda and J. A. Marco, Eur. J.
Org. Chem., 2014, 2284-2296. (c) C. Vilanova, S. Torijano-
Gutiérrez, S. Díaz-Oltra, J. Murga, E. Falomir, M. Carda and J.
A. Marco, Eur. J. Med. Chem., 2014, 87, 125-130. (d) S.
Torijano-Gutiérrez, C. Vilanova, S. Díaz-Oltra, J. Murga, E.
Falomir, M. Carda, M. Redondo-Horcajo, J. F. Díaz, I.
Barasoain and J. A. Marco, Arch. Pharm., 2015, 348, 541-547.
(e) L. Conesa-Milian, E. Falomir, J. Murga, M. Carda, E.
Meyen, S. Liekens and J. A. Marco, Eur. J. Med. Chem., 2018,
147, 183-193.
28 X. Wu, P. Nilsson and M. Larhed, J. Org. Chem., 2005, 70
,
346-349.
13 G. R. Pettit, S. B. Singh, M. R. Boyd, E. Hamel, R. K. Pettit, J.
M. Schmidt and F. Hogan, J. Med. Chem., 1995, 38, 1666-
1672.
14 (a) N. J. Lawrence, F. A. Ghani, L. A. Hepworth, J. A. Hadfield,
A. T. McGown and R. G. Pritchard, Synthesis, 1999, 1656-
1660. (b) J. E. Sheldon, M. M. Dcona, C. E. Lyons, J. C. Hackett
and M. C. T. Hartman, Org. Biomol. Chem., 2016, 14, 40-49.
(c) R. Gaspari, A. E. Prota, K. Bargsten, A. Cavalli, M. O.
Steinmetz, Chem., 2017,
15 (a) For a review on uses of silicon in medicinal chemistry,
see: A. K. Franz and S. O. Wilson, J. Med. Chem. 2013, 56
388-405. (b) For recent example of the use of
2, 102-113.
,
a
organogermanium compounds in medicine, see: J. Jiang, S.
Yao, H.-H. Cai, P.-H. Yang and J. Cai, Bioorg. Med. Chem. Lett.
2013, 23, 5727-5732. (c) For a review on tin-based antitumor
drugs, see: M. Gielen, Appl. Organometal. Chem. 2002, 16
481-494.
,
16 (a) A. T. McGown and B. W. Fox, Anti-cancer drug design,
1989, , 249-254. (b) R. R. Monaco, W. C. Gardiner and S.
3
Kirschner, Int. J. Quantum Chem., 1999, 71, 57-62. (c) C.
James, G. R. Pettit, O. F. Nielsen, V. S. Jayakumar and I. H.
Joe, Spectrochim. Acta Part A, 2008, 70, 1208-1216.
17 D. C. Harrowven, I. L. Guy, M. Howell and G. Packham,
Synlett 2006, 2977-2980.
18 W. H. Moser, Tetrahedron 2001, 57, 2065-2084.
19 (a) Group 13 elements: K. Yoshida, T. Furuyama, C. Wang, A.
Muranaka, D. Hashizume, S. Yasuike and M. Uchiyama, J.
Org. Chem., 2012, 77, 729-732; (b) group 14 elements: L. G.
Mercier, S. Furukawa, W. E. Piers, A. Wakamiya, S.
Yamaguchi, M. Parvez, R. W. Harrington and W. Clegg,
Organometallics, 2011, 30, 1719-1729; (c) group 15
elements: S. Yasuike, H. Ohta, S. Shiratori, J. Kurita and T.
Tsuchiya, J. Chem. Soc. Chem. Comm., 1993, 18, 1817-1819.
This journal is © The Royal Society of Chemistry 20
J. Name., 2013, 00, 1-3 | 13
Please do not adjust margins