Isocombretastatins a versus combretastatins a: the forgotten isoCA-4 isomer as a highly promising cytotoxic and antitubulin agent.
ABSTRACT Herein is reported a convergent synthesis of isocombretastatins A, a novel class of potent antitubulin agents. These compounds having a 1,1-diarylethylene scaffold constitute the simplest isomers of natural Z-combretastatins A that are easy to synthesize without need to control the Z-olefin geometry. The discovery of isoCA-4 with biological activities comparable to that of CA-4 represents a major progress in this field.
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ABSTRACT: Microtubules are intracellular organelles formed from the protein tubulin. These organelles have a number of essential cellular functions including chromosome segregation, the maintenance of cell shape, transport, motility, and organelle distribution. Drugs that affect the tubulin-microtubule equilibrium (taxol, vinca alkaloids) are effective anticancer drugs. This review describes the molecular target, methods used in screening, the structures of compounds known to interact with tubulin, and the clinical use of these agents. In addition the ability of these agents to destroy tumour vasculature is described. This represents an exciting new molecular target in the design of anticancer drugs.Progress in cell cycle research 02/2003; 5:309-25.
- Nature 03/1979; 277(5698):665-7. · 38.60 Impact Factor
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ABSTRACT: In this review, an attempt has been made to throw light on the mechanism of action of colchicine and its different analogs as anti-cancer agents. Colchicine interacts with tubulin and perturbs the assembly dynamics of microtubules. Though its use has been limited because of its toxicity, colchicine can still be used as a lead compound for the generation of potent anti-cancer drugs. Colchicine binds to tubulin in a poorly reversible manner with high activation energy. The binding interaction is favored entropically. In contrast, binding of its simple analogs AC or DAAC is enthalpically favored and commences with comparatively low activation energy. Colchicine-tubulin interaction, which is normally pH dependent, has been found to be independent of pH in the presence of microtubule-associated proteins, salts or upon cleavage of carboxy termini of tubulin. Biphasic kinetics of colchicines-tubulin interaction has been explained in light of the variation in the residues around the drug-binding site on beta-tubulin. Using the crystal structure of the tubulin-DAMAcolchicine complex, a detailed discussion on the pharmacophore concept that explains the variation of affinity for different colchicine site inhibitors (CSI) has been discussed.Medicinal Research Reviews 02/2008; 28(1):155-83. · 9.58 Impact Factor
pubs.acs.org/jmcPublished on Web 06/16/2009
r2009 American Chemical Society
4538J. Med. Chem. 2009, 52, 4538–4542
Isocombretastatins A versus Combretastatins A: The Forgotten isoCA-4 Isomer as a Highly Promising
Cytotoxic and Antitubulin Agent
Samir Messaoudi,†Bret Tr? eguier,†Abdallah Hamze,†Olivier Provot,†Jean-Franc -ois Peyrat,†Jordi Rodrigo De Losada,†
Jian-Miao Liu,‡J? er^ ome Bignon,‡Joanna Wdzieczak-Bakala,‡Sylviane Thoret,‡Joelle Dubois,‡Jean-Daniel Brion,†and
Mou^ ad Alami*,†
†Univ Paris-Sud, CNRS, BioCIS-UMR 8076, Laboratoire de Chimie Th? erapeutique, Facult? e de Pharmacie, 5 rue J.-B. Cl? ement,
Ch^ atenay-Malabry F-92296, France, and‡Institut de Chimie des Substances Naturelles (ICSN), UPR 2301 CNRS, Avenue de la Terrasse,
F-91198 Gif-sur-Yvette, France
Received March 25, 2009
Herein is reported a convergent synthesis of isocombretastatins A, a novel class of potent antitubulin
Z-combretastatins A that are easy to synthesize without need to control the Z-olefin geometry. The
discovery of isoCA-4 with biological activities comparable to that of CA-4 represents a major progress
in this field.
The discovery of natural substances capable of interfering
with the assembly or disassembly of microtubules has drawn
much attention because microtubules are recognized as an
attractive pharmacological target for anticancer drugs.1,2
Natural substancesthatareable tomodulate the microtubule
assembly can be broadly divided into two groups. In the first
microtubules, resulting in highly stable, nonfunctional as-
sembled microtubules.3In contrast to taxanes, the vinca
alkaloids (e.g.; vincristine, vinblastine, etc.), dolastatins as
these complex molecules have limitations resulting from high
toxicity, difficulty of synthesis, and some of them become
rapidly prone to resistance phenomena.5Among the large
class of natural substances known as inhibitors of tubulin
assembly, combretastatins A-1 to A-6, isolated from the
African willow tree Combretum caffrum, are currently the
simplest structures and have attracted the attention of many
medicinal chemists for the rational design of antitubulin
agents (Figure 1).6The cell growth inhibition of combretas-
of 60 human tumor cell lines.6cThe most potent member of
these natural stilbenes family is CA-4 (GI50=3.2 nM
CA-1 (GI50=16.2 nM) and CA-2 (GI50=31.6 nM), respec-
tively, were about one-fifth and one-tenth as potent as CA-4.
In another study,6bCA-3 was found to retain significant
growth cell inhibition against the murine P-388 lymphocytic
leukemia (ED50=26 nM), while CA-5 displayed a significant
CA-6 was essentially inactive. Furthermore, combretastatins
A-1 to A-4 were identified as potent inhibitors of tubulin
polymerization at the micromolar concentration (2-5 μM)
due to their ability to rapidly bind to tubulin at the colchicine
site. The recent interest in the anticancer potential of the
combretastatin A-series has gained momentum following the
observation that CA-4 and CA-1 induce rapid and reversible
an antivascular mechanism of action.7The water-soluble pro-
degeneration (AMD) or anaplastic thyroid cancer.9
The relative simplicity of the 1,2-diarylethylene scaffold of
combretastatin A-series, along with their biological proper-
ties, resulted in extensive structure-activity relationship
(SAR) studies.10In fact, over the past decade, the literature
for the last five years, more than 250 publications and 170
patents concern combretastatins, many of which show the
In spite of these SAR studies and the impressive number of
analogues synthesized (>28000), only the CA-4 and CA-1
remain the most promising molecules as their prodrugs, CA-
4P and CA-1P, have a significant impact on the clinical
management of cancer.9
Despite their remarkable therapeutic interest, these
Z-natural stilbene compounds are prone to double-bond
isomerization during storage and administration, leading to
the E-isomer, which dramatically displayed a reduced inhibi-
noted that the third isomer of the combretastatin A-series,
named isocombretastatins A (isoCA), has never been stu-
died despite the impressive number of synthetic analogues.
*To whom correspondence should be addressed. Phone: 33(0)
22.214.171.124.87. Fax: 33(0)126.96.36.199.28. E-mail: mouad.alami@u-psud.
concentration; ED50, the half maximal effective dose concentration).
Brief ArticleJournal of Medicinal Chemistry, 2009, Vol. 52, No. 144539
attractive class of compounds for cancer treatment.
In our efforts to discover novel tubulin assembly inhibi-
tors,13we hypothesized that isocombretastatins A with 1,1-
diarylethylene scaffold could be as active as those of natural
combretastatins A. Here, we report on synthesis of isocom-
bretastatins A-1 to A-5 (Figure 1) and the evaluation of their
biological properties. This study would bring additional
information on the importance of the carbonyl oxygen of
phenstatin to form crucial interactions at the colchicine site
isoCA were examined for their capacity to inhibit cancer cell
growth and to act as potential antimitotic agents.
the presence of Pd2(dba)3and Xphos provided the resulting
coupling product, which was subsequently desilylated under
alkaline media to obtain the target isoCA-4 (Scheme 1). In
the same way, starting from hydrazones 3, 7, and 9, the
coupling with appropriate aryl halides 4, 5, and 10 furnished
good yield (Scheme 1). It should be noted that isoCA-5 is
either the isomer of position of the CA-5 or its trans isomer
Results and Discussion
The isocombretastatins A-1 to A-5 were evaluated for their
cytotoxic activities against four types of human cell lines,
human colon carcinoma HCT116 cells, chronic myelogenous
cells, and human breast cancer MDA-MB231 cells. The
the newly synthesized isoCA-4 exhibited approximatively a
10-fold greater cytotoxic activity (nanomolar level) than that
of phenstatin and colchicine on the four tested cell lines.
Interestingly, isoCA-4 activity against the above cell lines is
comparable to the natural CA-415isomer (2-5 nM). These
resultssuggestthatswitchingan aromaticring from the C2to
the C1 position retained potent cytotoxicity and that, in this
series, the 1,1-ethylene bridge would be regarded as a bioisos-
tere16of the Z-1,2-ethylene one. Next, other isocombretasta-
tins A were examined. Again, isoCA-2 and isoCA-3 showed
respectively), which are comparable to their CA-2 and CA-3
parents.6Inverting a MeO substituent by a OH group on
the aromatics led to a dramatic reduction in cytotoxicity
when isoCA-4 and isoCA-5 were compared. The isoCA-5
Figure 1. Combretastatins A, synthetic tubulin assembly inhibitor phenstatin (1), and structures of the synthesized isocombretastatins A-1 to
aReagents and conditions: (a) TsNHNH2, EtOH, reflux. (b) ArX,
Pd2(dba)3, Xphos, tBuOLi, dioxane, 70 ?C. (c) K2CO3, MeOH. (d) (i)
n-BuLi, THF, -78 ?C then MeCHO; (ii) PCC, CH2Cl2, 20 ?C.
Table 1. Cytotoxicity of isoCA on the Human Cancer Cell Lines and
aIC50is the concentration of compoundneededto reducecell growth
by 50% following 72 h cell treatment with the tested drug (average of
three experiments).bITP=Inhibition of tubulin polymerization; IC50is
the concentration of compound required to inhibit 50% of the rate of
microtubule assembly (average of three experiments).cNonactive.
4540Journal of Medicinal Chemistry, 2009, Vol. 52, No. 14Messaoudi et al.
but is 1000-fold less important than isoCA-4.
As combretastatin A-series have been well documented to
interact with tubulin, isocombretastatins A-1 to A-5 were
evaluated for their antitubulin assembly activities (Table 1).
natural isomer CA-4 and isoCA-4, the latter showed an
(2.2 μM) than that exhibited by CA-4 (1.0 μM) or colchicine
(2.1 μM). More interestingly, the activity of isoCA-4 is
comparable with that of phenstatin (2.0 μM) despite the
absence of the carbonyl function that was suggested to play
cytotoxic isoCA-1 (2.1 μM).
The effects of the most active agent isoCA-4 on cell cycle
distribution were analyzed in K562 cells cultured for 24 h in
the presence of increasing amounts of isoCA-4. The results
presented in Figure 2 demonstrate a dose-dependent increase
that isoCA-4 induces selectively arrested cell division in the
G2/M phase of the cell cycle.
The X-ray crystal structure of isoCA-4 depicted in
Figure 3A shows that the conformation of the 1,1-diarylethy-
lene scaffold is not planar and the planes of the two aromatic
ringsare inclined toward each other (dihedral angle=68?).A
similar nonplanarity was observed in the case of the natural
substance CA-4.18It seems that in combretastatin A-series,
position of the ethylene bridge preserves the spatial relationship
of the two aromatic rings (cis-relationship) in the 1,1-diaryl-
activities exhibited by these substances.
Adopting the conformation found in its X-ray crystal
structure, isoCA-4was computer dockedinsidethecolchicine
binding site. For this purpose, the X-ray structure of tubulin-
colchicine complex (Code PDB: 1sa0)19was used. Figure 3B
shows the docking-derived superimposition of isoCA-4, CA-
4, phenstatin, and colchicine (blue, green, red, and orange,
respectively). As expected, isoCA-4, CA-4, and phenstatin
show a nice fitting between them and the colchicine X-ray
structure into the binding site (Figure 3B). As previously
Figure 3. (A) X-ray crystal structure of isoCA-4. (B) Putative binding mode of isoCA-4 (blue), CA-4 (green), phenstatin (red), and colchicine
(orange) in the colchicine binding site.
Figure 2. Effect of isoCA-4 on cell-cycle distribution in chronic
myelogenous leukemia K562 cells determined by flow cytometry
analysis. DNA content was assessed via propidium iodide staining.
Figure 4. IsoCA-4 analogues 12 and 14 with a tri- and tetra-substituted double bonds.
Brief ArticleJournal of Medicinal Chemistry, 2009, Vol. 52, No. 14 4541
also well fitted. Hydroxyl groups belong to CA-4, isoCA-4,
and phenstatin show a hydrogen bond with backbone of
Val181 as proposed by Nguyen et al.17
Because the 1,1-ethylene bridge appears to be a suitable
bioisosteric replacement for the Z-1,2-ethylene one in this
series, we attempted to extend this finding to other potent
antitubulin agents. We anticipated that substances 12 and 14
with a one-carbon sp2bridge would be as active as their
known synthetic parent compounds 1120and 1321having a
tri- or tetra-substituted double-bond, respectively (Figure 4).
To this end, the synthetic strategy that allowed the prepara-
tion of 12 and 14 utilized a Wittig olefination of phenstatin
silyl ether intermediate12with the appropriate ylides followed
evaluated for their cytotoxic effects against HCT116 cell lines
as reference compound (Figure 4). Diarylacrylonitrile 12
related to CC-507922was tested as a mixture of E and Z
isomers (1/1) and was found to exhibit a high activity with an
IC50of 3.0 nM (IC50= 2.3 nM for CA-4) and difluorinated
substance 14 showed almost the same activity as the parent
compound 13 (H460 nonsmall cell lung carcinoma). Interest-
ingly, 12 and 14 displayed strong antitubulin activities with
IC50of 4.3 and 2.5 μM, respectively. These results clearly
demonstrate that the modification made on the ethylene
bridge maintains cytotoxic and antitubulin polymeri-
zation activities and, consequently, validate our bioisosteric
replacements wherein a 1,1-ethylene moiety mimics a Z-1,2-
In the present study, we have described the synthesis and
tastatins A, with strong anticancer activities. These com-
pounds, in contrast to their parent 1,2-diarylethylene
derivatives, are easy to synthesize without the need to control
the olefin geometry and constitute the simplest isomers of
natural combretastatins A. The most active agent, isoCA-4,
shares a striking structural similarity with phenstatin and
displayed a 10-fold better cytotoxic activity (2 nM). More-
over, isoCA-4 appears to elicit its cytotoxicity in a fashion
similar to CA-4, via inhibition of tubulin polymerization,
which then leads to cell cycle arrest in G2/M.1
As the replacement of the 1,2-ethylene by the 1,1-ethylene
bridge resulted in retention of biological activities, our results
may encourage the use of this scaffold in future structure-
activity relationship studies. All of this data make these
compounds worthy of further in vitro and in vivo investiga-
findings is currently underway; the results of synthetic and
biological studies will be reported in due course.
Procedure for the synthesis of isoCA-1 to isoCA-5. To a
dioxane (6 mL) solution of N-tosylhydrazone (0.42 mmol),
tBuOLi (84 mg, 1.05 mmol), Pd2dba3(44 mg, 0.042 mmol),
and X-Phos (40 mg, 0.084 mmol) was added the aryl halide
(0.42 mmol) in dioxane (1 mL). The mixture was stirred at 75 ?C
filtered over a pad of celite. After concentration, the residue
was dissolved in MeOH (3 mL), K2CO3(116.0 mg, 0.84 mmol)
added and the aqueous phase was extracted with Et2O (3 ?
10mL). The organic phasewas washed withbrine(15 mL), dried
over MgSO4, and concentrated in vacuo to give a residue which
was purified by silica gel chromatography to yield isoCA. Purity
was determined by elemental analysis and HPLC; purity of key
target compounds isoCA-1 to isoCA-5 was >98%.
Acknowledgment. We thank the CNRSfor support of this
research and the ICSN for a doctoral fellowship to B.T.
Supporting Information Available: Typical experimental pro-
cedure, analytical data, crystallographic information files (CIF),
modeling programs,1H,13C NMR, and MS data of new com-
pounds. This material is available free of charge via the Internet
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