Synthesis and biological evaluation of combretastatin analogs as cell cycle inhibitors of the G1 to S transition in Saccharomyces cerevisiae

Article abstract of DOI:10.1016/j.bmcl.2010.03.066

A series of Z and E combretastatin A-4 analogs bearing different substituents (OH, F, NO₂, NH₂, B(OH)₂) in the 3′ position were synthesized. These derivatives and Z and E combretastatin A-1 were analysed by monitoring their ability to inhibit cell growth in Saccharomyces cerevisiae. Combretastatin A-1 (2a), A-4 (2b) and compound 2c were found to inhibit yeast growth. Moreover, combretatstatin A-4 (2b) and compound 2c induced a G1 arrest by affecting the synthesis of Clb5 protein, the principal S-phase cyclin. The G1 arrest is coincident with the activation of the stress activated kinase Snf1.

Full text of DOI:10.1016/j.bmcl.2010.03.066

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2780–2784
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of combretastatin analogs as cell cycle
inhibitors of the G1 to S transition in Saccharomyces cerevisiae
b
b
a
b
Paola Coccetti a, , Giuseppe Montano , Alessandro Lombardo , Farida Tripodi , Fulvia Orsini ,
*
Roberto Pagliarin b
a Dipartimento di Biotecnologie e Bioscienze, Università Milano-Bicocca, P.zza della Scienza, 2, 20126 Milano, Italy
b Dipartimento di Chimica Organica e Industriale, Università di Milano, Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of Z and E combretastatin A-4 analogs bearing different substituents (OH, F, NO2, NH2, B(OH)2) in
the 30 position were synthesized. These derivatives and Z and E combretastatin A-1 were analysed by
monitoring their ability to inhibit cell growth in Saccharomyces cerevisiae. Combretastatin A-1 (2a), A-4
(2b) and compound 2c were found to inhibit yeast growth. Moreover, combretatstatin A-4 (2b) and com-
pound 2c induced a G1 arrest by affecting the synthesis of Clb5 protein, the principal S-phase cyclin. The
G1 arrest is coincident with the activation of the stress activated kinase Snf1.
Received 2 January 2010
Revised 12 March 2010
Accepted 13 March 2010
Available online 17 March 2010
Keywords:
Cell cycle
Ó 2010 Elsevier Ltd. All rights reserved.
Saccharomyces cerevisiae
G1/S transition
Combretastatins
The combretastatins were originally isolated from the South
African tree Combretum caffrum.1 Combretastatin A-4 (CA4) is a
natural cis-stilbene used in traditional medicine for the treatment
of hepatitis and malaria.2,3 CA4 is a microtubule-destabilizing
agent that inhibits microtubule assembly by binding to tubulin,
similar to the well-known microtubule-targeted agent colchicine.4
CA4P, the water soluble prodrug, induces microtubule disassembly
and leads to a dramatic cytotoxicity in proliferating vascular endo-
thelial cells which show a strong inhibition of malignant hemangio-
endothelioma cell proliferation.5,6 In experimental tumors, anti-
vascular effects are observed within minutes of CA4P administration
which rapidly leads to extensive ischemic necrosis in areas that are
often resistantto conventionalanti-cancertreatments.5,7–10 In keep-
ing with their tumor vascular disrupting activity at well tolerated
doses,11,12 combretastatin A-4-P and combretastatin A-1-P (the
prodrug of the natural parent CA1) are now collectively classified
as vascular disrupting agents (VDAs), since they cause rapid shut
down of the established tumor vasculature.13 CA4P, which is the
most widely studied VDA of the microtubule depolymerising family,
is being evaluated in Phase III clinical trials for cancer treatment.13
CA1P, which is an even more potent VDA than CA4P,14 is also now
being clinically tested.13
tent and leads to the death of mitotic cells. The mitotic arrest is
associated with elevated levels of cyclin B1 and of p34cdc2 activity.5
Although most previous studies have focused on the anti-vascu-
lar effect of CA4P and used the endothelial cells as major target
cells, it has also been suggested that the anti-tumor effect of
CA4P may be an integrated consequence of the destruction of tu-
mor vascular network and direct killing of tumor cells. In fact, it
was shown that CA4P has primary anti-neoplastic activity against
human anaplastic thyroid carcinoma cell lines and xenograft
tumors.15
Recently, it has been demonstrated that CA4 stimulates both
extracellular signal-regulated kinases (ERK1/2) and p38 MAPK in
human hepatocellular carcinoma cell lines and that only the inhi-
bition of p38 MAPK synergistically enhances the cytotoxicity of
CA4,16 providing novel evidence that the combination of p38 MAPK
inhibitors with CA4 may represent a promising strategy for cancer
therapy. In addition, it was also shown that CA4 inhibits growth in
several human gastric cancer cell lines and, more interestingly, the
inhibition of the oncogenic pathway of AKT by CA4 results in re-
duced metastasis.17
Further investigation identifies CA4 as an activator of AMP-acti-
vated protein kinase (AMPK),18 which is a key regulator of energy
balance involved in response to cellular stress in mammalian
cells.19 It is known that activation of AMPK, through the up-regu-
lation of p53, increases the level of the Cdk1 inhibitor p21CIP, which
in turn induces a G1 cell cycle arrest.20
CA4P addition to proliferating endothelial cells damages mitotic
spindles, arrests cells at metaphase with a post-synthetic DNA con-
Although the anti-fungal activity of combretastatins has been
* Corresponding author. Tel.: +39 02 64483521; fax: +39 02 64483519.
E-mail address: paola.coccetti@unimib.it (P. Coccetti).
previously reported,21,22 the effects on yeast cell cycle progression
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.066

P. Coccetti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2780–2784
2781
have not yet been evaluated. One of the best analysed organisms
for cell cycle studies is the budding yeast Saccharomyces cerevisiae,
which can grow on synthetic media at widely different growth
rates. It is also well known at both the genetic and biochemical le-
vel, being the first eukaryote for which the complete genome se-
quence was obtained.23 Genome-wide functional analyses,
including the analysis of transcriptional responses to a range of
stimuli, and comprehensive maps of protein–protein interactions
further facilitate the study of the yeast cell cycle.24,25 Finally, the
high degree of conservation with mammalian cells in terms of gene
sequences, proteins and biochemical functions makes S. cerevisiae
very appealing for anti-cancer drug discovery.26 In budding yeast,
the cell cycle begins with the synthesis of the initiator cyclin
Cln3, which binds to and activates the principal cyclin-dependent
kinase Cdk1.27 Substrate of the Cln3/Cdk1 complex is the transcrip-
tional inhibitor Whi5 which, upon phosphorylation, is translocated
out of the nucleus releasing the SBF and MBF transcription fac-
tors.28,29 SBF and MBF transcription factors promote the expression
of the other G1 cyclins, Cln1 and Cln2, responsible for bud forma-
tion and spindle pole body duplication,27,30 and of the S-phase cyc-
lins Clb5 and Clb6.31 In G1 phase, Clb5,6/Cdk1 complexes are
inhibited by the Cdk inhibitor Sic1 which prevents premature
DNA replication.32 This inhibition is removed following phosphor-
ylation of Sic1 by Cln1,2/Cdk1 complexes, promoting its degrada-
tion via an ubiquitin-mediated mechanism, allowing Clb5,6/
Cdk1-dependent onset of DNA replication.33,34
pounds 2e and 3e were further submitted to a reduction reaction,41
using sodium dithionite as reducing agent; the amino derivatives 2f
and 3f were obtained with 60% yield after column purification. The
boronic acid bioisostere 2c was presented by Kong et al.37 as a final
product of two reactions performed in anhydrous conditions and the
fluoro derivative 2dwas synthesizedby Lawrence et al.38 with a Wit-
tig coupling at ꢀ78 °C. All compounds 2(b–e) and 3(b–e) were pre-
pared in a single step from the corresponding aldehyde and
phosphonium salt without dry solvents or low-temperature
conditions.
Exponentially growing cells of BY4741 S. cerevisiae strain (Sup-
plementary data Table 2) were then grown in complete medium
containing 2% glucose and at time 0 compounds shown in Scheme
1 (2a–f and 3a–f) were added at a final concentration of 150, 300,
and 450 lM. Samples were taken at the indicated time points to
determine cell growth, budding index and DNA content by FACS
analysis (Fig. 1A and B). Compounds 2d–f and 3a–f and compounds
3a, 3b and 3c had no effect on yeast growth at all tested concentra-
tions (data not shown). On the contrary, when log-phase cultures
were grown and treated with molecules 2a, 2b, and 2c, cell growth
arrested in a dose-dependent manner (Fig. 1A). Remarkably, some
differences were observed among the behaviour of the various
compounds: molecule 2a caused a small increase of budding index
in comparison to the control culture, the majority of the population
being arrested with a post-synthetic DNA content (Fig. 1A and B)
and with short spindles typical of cells blocked in metaphase (data
not shown). On the other hand, compounds 2b and 2c induced a
With the goal of elucidating the effects of combretastatins on
cell cycle progression, the in vivo activity of compounds 2(a–f)
and 3(a–f) was analysed by monitoring their ability to inhibit yeast
cell growth. We synthesized the CA1 (2a, 3a), the CA4 (2b, 3b), and
the eight CA4 derivatives 2(c–f) and 3(c–f), where the hydroxy
group of the natural compound was replaced by fluoro, nitro, ami-
no, and boronic acid moieties, either as Z or E isomer. The strategy
used to prepare the cis and trans analogs is outlined in Scheme 1.
CA1 (2a, 3a) was obtained according to a methodology described in
the literature,35 whereas the eight derivatives 2(b–e) and 3(b–e)
were prepared following a protocol alternative to those reported
in the literature. All compounds are already known in the litera-
ture36–39 but we developed a clean and more convenient methodol-
ogy for their synthesis, which allowed us to skip the use of moisture-
sensitive reagents and critical reaction conditions. We thus synthe-
sized compounds 2(b–e) and 3(b–e) by a transfer-phase Wittig cou-
pling of p-methoxybenzaldehyde bearing in position 30 a boronic
moiety, a fluoro atom or a nitro group, respectively, and tri-
phenyl(3,4,5-trimethoxybenzyl)phosphonium chloride.40 The most
versatile reaction conditions were dichloromethane and a water
solution saturated with potassium hydrogen carbonate. The cis/
trans mixtures were purified and isomers were separated by flash
chromatography in a gradient of acetone/petroleum ether. The com-
strong reduction of budding index (42% with 450 lM, compare
with 70% of the untreated culture, Fig. 1A) and about 50% of the
population was completely arrested in G1 phase after 3 h treat-
ment (Fig. 1B). In order to understand the molecular mechanism
that accounts for G1 arrest mediated by compounds 2b and 2c,
we analysed the pattern of expression of the main regulators of
G1 phase, the cyclins Cln1, Cln2 and Clb5 and the inhibitor Sic1,
following treatment with the drugs. To this end, strains bearing a
TAP-tagged version of Cln1, Cln2, Clb5, and Sic1 were grown until
exponential phase and treated with 450 lM of compounds 2b and
2c as reported above. Western blot analysis of samples taken after
3 h treatment showed that combretastatins 2b and 2c downregu-
lated the level of Clb5 cyclin, which was required for the onset of
DNA replication (Fig 1C). Cln1, Cln2, and Sic1 were still present
in G1 arrested cells (Fig. 1C).
To further investigate whether the down-regulation of Clb5
expression was dependent on the presence of compounds 2b and
2c, exponentially growing Clb5-TAP strain was synchronized in
G1 phase by
a-factor treatment and released in fresh medium in
the presence or absence of combretastatin 2b or 2c. As expected,
control cells started to enter S-phase 20–30 min after the release,
in keeping with the increase of Clb5 level (Fig. 2A and B). In con-
trast, Clb5 was not detectable in cells treated with both com-
pounds 2b and 2c and, consistently, DNA replication did not
begin (Fig. 2A and B).
Taken together these data suggest that combretastatins 2b and
2c determine a G1 arrest due to a decreased level of Clb5, which is
not sufficient to start DNA replication. This shows a new block of
yeast cell cycle which has not previously been identified in mam-
malian cells.
Since CA4 is known to induce AMPK activation in several mam-
malian cell lines,18 we analysed the effect of compounds 2b and 2c
on the activation of Snf1, the yeast orthologue of AMPK.42,43 Snf1
plays an important role in regulating a wide range of responses
to stress caused by nutrient limitation and salt stress43,44 and
phosphorylation on threonine 210 (T210), by one of the redundant
upstream kinases Sak1, Tos3 or Elm1, is required for its activa-
tion.45,46 We then tested whether Snf1 became phosphorylated at
T210 following 2b and 2c treatment. Exponentially growing cells
OMe
R
MeO
MeO
MeO
OMe
OMe
MeO
OMe
CHO
2
R'
PPh Cl
3
R'
+
i
R
MeO
MeO
1
OMe
OMe
3
R'
R
ii
i
2a (CA1) + 3a
2b (CA4) + 3b
2c + 3c
1a (R=OH;
1b (R =OH;
1c (R=B(OH)2
1d (R =F;
R' = OH)
R'= H)
R' = H)
R' =H)
R'= H)
i
;
i
2d
2e
+
+
3d
3e
iii
i
1e (R=NO2;
(2f + 3f ), f (R=NH2 ; R'= H)
Scheme 1. Reagents and conditions: (i) KHCO3/H2O/CH2Cl2, rt; (ii) (1) TBDMSCl/
DIPEA/DMF; (2) BuLi/THF, from ꢀ20 °C to rt; (3) TBAF, THF; (iii) Na2S2O4, acetone/
water 2:1, 50 °C.

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P. Coccetti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2780–2784
A
2a
100%
1,20E+07
1,00E+07
8,00E+06
6,00E+06
4,00E+06
2,00E+06
0,00E+00
Control
80%
60%
150uM 2a
300uM 2a
450uM 2a
Control
40%
150uM 2a
300uM 2a
20%
450uM 2a
0%
0
30
60
90
120
150
180
0
30 60
90
120
150
180
time (min)
time (min)
2b
100%
80%
60%
40%
20%
0%
2,50E+07
2,00E+07
1,50E+07
1,00E+07
5,00E+06
0,00E+00
Control
150uM 2b
300uM 2b
450uM 2b
Control
150uM 2b
300uM 2b
450uM 2b
0
30
60
90
120
150
180
0
30
60
90
120
150
180
time (min)
time (min)
2c
3,00E+06
2,50E+06
2,00E+06
1,50E+06
1,00E+06
5,00E+05
0,00E+00
100%
80%
60%
40%
20%
0%
Control
150uM 2c
300uM 2c
450uM 2c
Control
150uM 2c
300uM 2c
450uM 2c
0
30
60
90
120
150
180
0
30
60
90
120
150
180
time (min)
time (min)
B
Control
+2a
+2b
+2c
C
-
+
2b
-
+
2c
Cln1-TAP
Pgk
Cln1-TAP
Pgk
Cln2-TAP
Pgk
Cln2-TAP
Pgk
Clb5-TAP
Pgk
Clb5-TAP
Pgk
Sic1-TAP
Pgk
Sic1-TAP
Pgk
Figure 1. Combretatstatin 2a, 2b and 2c inhibit the growth of budding yeast. Exponentially growing cells of BY4741 strain in YP medium, supplemented with 2% glucose,
were treated with combretastatin 2a, 2b and 2c (150 M, 300 M and 450 M) at 25 °C. Samples were taken at the indicated time points to assay (A) cell density and budding
l
l
l
index; (B) DNA content by FACS analysis. (C) Protein levels by western blot analysis of TAP-tagged proteins from strains treated for 3 h with 450
lM of compounds 2b and 2c.
Anti-TAP antibody was used and Pgk1 was used as a loading control.

P. Coccetti et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2780–2784
2783
A
Control
+ 2b
+ 2c
100’
80’
60’
50’
40’
30’
20’
10’
0
100’
80’
60’
50’
40’
30’
20’
10’
0
100’
80’
60’
50’
40’
30’
20’
10’
0
Cyc
Cyc
Cyc
Time (min)
Time (min)
Time (min)
B
Clb5
Pgk
Figure 2. G1 arrested cells treated with combretastatin 2b and 2c are unable to traverse the G1 phase of the cell cycle. Clb5-TAP strain was synchronized in G1 phase by
addition of -factor treatment, then washed and released in fresh medium containing compounds 2b and 2c (450 M); in control culture only DMSO was added. Samples
a
l
were taken at the indicated time points to assay (A) DNA content by FACS analysis; (B) Clb5 level by western blot analysis using anti-TAP antibody. Pgk1 was used as a loading
control.
of a yeast strain expressing Snf1-9myc were treated with com-
pounds 2b and 2c for 3 h. Western blot analysis with specific
anti-P-Thr-210 antibody revealed a very low level of Snf1 phos-
phorylation in the presence of combretastatin 2c, while compound
2b caused a consistent T210 phosphorylation on Snf1, comparable
to that observed when cells were grown in low glucose concentra-
tion (0.05%), a known Snf1-activating condition (Fig. 3).47
malian cells—AMPK kinase—regulates cell proliferation by per-
forming an energy-checkpoint at G1/S phase by the accumulation
of p53 and of the cyclin-dependent kinase inhibitors, p21 and
p27.48 Another Snf1 homologue—DmAMPK in Drosophila melano-
gaster—was also found to be involved in cell cycle arrest, at the
G1/S boundary, resulting in reduced levels of the rate-limiting pro-
tein Cyclin E.49
Results reported in this Letter newly indicate an involvement of
combretastatin 2b and 2c in arresting yeast growth inducing a G1
block not previously identified in mammalian cells, since they pre-
vent DNA replication by affecting the synthesis of Clb5 protein, the
principal S-phase cyclin. Furthermore, the G1 arrest is coincident
with the activation of the stress activated kinase Snf1 in the case
of compound 2b and to a lesser extent with combretastatin 2c. It
has previously been reported that the homologue of Snf1 in mam-
Considering that CA4 has been identified as an activator of
AMPK in several mammalian cell lines18 and that data presented
in this Letter show a similar role for Snf1, a full understanding of
Snf1/AMPK signalling pathways could be useful for the develop-
ment of optimal drugs for various diseases, since the AMPK path-
way is deregulated in several cancer types.
Furthermore, the novel results presented here underline once
more the importance of budding yeast as a model organism for
understanding how intracellular and extracellular signals are
transmitted to cell cycle machinery. These studies are of great
importance considering that proliferative disorders are now such
a major challenge for human health.
Acknowledgments
WB:Snf1-pT210
We thank Stefan Hohmann for providing anti-phospho-T210
Snf1 antiserum, Lilia Alberghina for constructive comments and
suggestions on the manuscript and Neil Campbell for language
checking.
This work has been supported by Grants from MIUR to P.C. (FAR
2007 and FAR 2008) and to F.O. (PRIN 2007, prot. 2007K29W5J).
WB:Snf1
IP anti-Snf1
Figure 3. Snf1 is phosphorylated on Thr210 in cells treated with combretatstatin
2b. Exponentially growing cells of Snf1-9myc strain were treated with 450 lM of
combretastatin A4 2b and 2c at 25 °C. In the control culture only DMSO was added
(lane 1, cells grown in 2% glucose). After 3 h samples were taken and Snf1-9myc
Supplementary data
was immunopurified from 500 lg of total protein extract with anti-myc antibody.
Western blot analysis to detect Snf1 phosphorylation on Thr210 was made with
anti-Thr210P antibody and with anti-myc antibody to detect Snf1 total level.
Supplementary data (synthesis, spectral data description, Table 2
of yeast strains used and yeast experiments performed) associated

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