Synthesis and antitumor-evaluation of cyclopropyl-containing combretastatin analogs

Article abstract of DOI:10.1016/j.bmcl.2009.10.064

Several derivatives of combretastatin have been prepared bearing a cyclopropyl unit instead of the natural occurring cis-double bond. Final products and synthetic intermediates were evaluated for their cytotoxic properties in two human cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2009.10.064

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6948–6951
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antitumor-evaluation of cyclopropyl-containing
combretastatin analogs
Rita Fürst a, István Zupkó b, Ágnes Berényi b, Gerhard F. Ecker c, Uwe Rinner a,
*
a Department of Organic Chemistry, University of Vienna, Währinger Straße 38, 1090 Vienna, Austria
b Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u 6, 6720 Szeged, Hungary
c Department of Medicinal Chemistry, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Several derivatives of combretastatin have been prepared bearing a cyclopropyl unit instead of the nat-
ural occurring cis-double bond. Final products and synthetic intermediates were evaluated for their cyto-
toxic properties in two human cancer cell lines.
Received 13 August 2009
Revised 14 October 2009
Accepted 14 October 2009
Available online 20 October 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Dedicated to Professor Johann Mulzer on the
occasion of his 65th birthday in recognition
of his contributions to synthetic organic
chemistry
Keywords:
Combretastatin
Tubulin binding
Anticancer drug
Colchicine
Stilbene
In 1982, Pettit and co-workers isolated highly oxygenated stil-
bene derivatives from the bark of the African willow tree Combre-
tum caffrum.1 The newly isolated compounds were named
combretastatins and were identified as natural products with
remarkable biological properties. Combretastatin A4 (1, Fig. 1), is
among the most potent cytotoxic compounds known to date.2,3
Combretastatins induce apoptotic cell death by selectively binding
to tubulin at the colchicine binding site resulting in disruption of
the formation of microtubules and cell cycle arrest at the transition
of meta- to ana-phase.4,5
Derivatives of combretastatin used in clinical studies are shown
in Figure 1.13–17 Although these compounds express high levels of
in vitro activity, the in vivo activity is limited because of the high
tendency of the system to undergo cis/trans isomerization. Several
publications describe the synthesis and biological evaluation of
derivatives with heterocyclic moieties instead of the cis-stilbene
unit.18–21 However, the overall polarity of the compounds is
strongly modified by such an electronic modification. One way to
bypass this problem is to incorporate a structural motif which
Since their discovery in 1982, numerous derivatives of combre-
tastatin have been prepared and subjected to biological activity
testing.6–10 As indicated in Figure 2, structural modifications are
generally possible either at the aromatic moieties (structural motif
A and C) or the two atom bridge connecting the aromatic rings
(structural motif B). Structure activity relationship studies revealed
that the methoxy substituents at ring A are required for biological
activity. Furthermore, free hydroxyl functionalities or other equiv-
alent hydrogen bonding donors at ring C and the cis-stilbene moi-
ety are essential for high levels of cytotoxicity.11,12
MeO
MeO
OMe
OH
OMe
Combretastatin A4 (CA4; 1)
MeO
MeO
MeO
MeO
O
OMe
OMe
OPO3Na2
N
H
OH
NH2HCl
OMe
Combretastatin A4 phosphate (CA4-P;
OMe
2
3
AC-7700 ( )
)
* Corresponding author. Tel.: +43 1 4277 52108; fax: +43 1 4277 9521.
E-mail address: uwe.rinner@univie.ac.at (U. Rinner).
Figure 1. Combretastatin A4 (1) and derivatives used in clinical studies.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.064

R. Fürst et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6948–6951
6949
and cyclohexene in 80% yield.24 Deprotection of the MOM ether
afforded the natural product (1) which was further converted to
phosphate 2 by treatment with dibenzyl phosphite and subsequent
cleavage of the benzyl groups with TMSBr.25 MOM-protected stil-
bene derivative 11 was used in the cyclopropanation reaction with
CH2N2 and Pd(OAc)2. Subsequent deprotection afforded the cyclo-
propyl-containing analog of combretastatin (4). Following the reac-
tion sequence described above, introduction of the phosphate group
for higher water solubility concluded the synthesis with the isola-
tion of 15 (Scheme 1).
B
MeO
MeO
MeO
MeO
A
C
OMe
OMe
OH
OH
OMe
CA4 (1)
OMe
Cyclopropyl derivative of CA4 (4)
Figure 2. Comparison of CA4 (1) and the cyclopropyl derivative of the natural
product (4).
The preparation of amine 23 and amide 25 is carried out in close
analogy. (Scheme 2). Sonogashira coupling26 of iodide 16 and
alkyne 6 followed by hydrogenation under Lindlar conditions using
Pd on CaCO3 afforded amine 19 in 83% yield. For reference purposes,
AC-7700 was prepared by benzotriazole promoted coupling of
amine 19 with Fmoc-protected serine acetate, basic cleavage of
the protecting groups (20) and precipitation of the hydrochloride.10
Cyclopropanation of free amine 19 could not be carried out suc-
cessfully. The problem was solved by introduction of a Boc group
on the nitrogen (21). Treatment of carbamate 21 with CH2N2 and
a catalytic amount of Pd(OAc)2 afforded cyclopropyl derivative 22
in 58% yield. Cleavage of the Boc group (23) followed by installa-
tion of the serine side chain as described above allowed the isola-
tion of the desired cyclopropyl derivative of AC-7700 (25).
All compounds reported herein were evaluated for in vitro cyto-
toxicity in HeLa (cervical adenocarcinoma) and MCF-7 (breast ade-
nocarcinoma)—using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide] assay.27 Combretastatin A4 and
other derivatives already used in clinical trials were included as
control substances. Results of this biological activity study are
summarized in Table 1.
maintains the overall polarity of the natural product and ensures
binding of the natural product derivative to tubulin but at the same
time locks the cis isomer of the two atom bridge. A possibility to
achieve this goal is to incorporate a fully saturated carbocycle in-
stead of the stilbene moiety. In addition to the advantage of the
fixed cis-relationship of the aromatic rings, variation of the ring
size allows fine-tuning of the angle and exact position of the aro-
matic portions.
Herein, we describe the synthesis and biological evaluation of
novel derivatives of combretastatin with cyclopropyl units instead
of the cis-double bond. All compounds containing the cyclopropyl
moiety were synthesized as racemates as we were mainly inter-
ested in investigating the effect of the angle and spatial arrange-
ment of the aromatic rings.
The synthesis of the natural product and the cyclopropyl deriva-
tive is shown in Scheme 1. The reaction sequence started by conver-
sion of the carbonyl moiety of trimethoxy benzaldehyde (5) into the
corresponding alkyne functionality with TMS-diazomethane.22 Al-
kyne 6 was coupled under Suzuki conditions with aromatic bromide
8,23 available by MOM-protection of phenol 7, to afford bis-function-
alized alkyne 9 in good yield. For biological activity studies, small
amounts of this material were deprotected to afford phenol 10 in
90% yield. Selective reduction of the triple bond in 9 to the cis-stil-
bene motif was achieved by hydroboration with BH3.THF complex
Alkyne derivatives obtained as intermediates in the synthesis of
the cyclopropane containing substrates were also employed in bio-
logical activity studies. As expected, these substrates were found
to be completely inactive and these findings confirm previous
studies.28
Combretastatin A4 and derivatives containing the cis-stilbene
moiety were found to be most active and these compounds express
CHO
MeO
MeO
OH
OMe
a
MeO
OMe
MeO
OMe
I
10
9
MeO
MeO
OMe
6
Br
MeO
MeO
MeO
NHR
OMe
OMe
5
a
d
+
Br
OMOM
OMe
NH2 MeO
OMe
b
c
17
18
R = H
R = Boc
g
MeO
OMe
OMe
6
f
16
OH
OMOM
MeO
MeO
OMe
7
OMe
8
e
b
MeO
MeO
MeO
MeO
MeO
MeO
f, g
MeO
O
P
OMe
OMe
OMe
OMe
NHR
NHBoc
O
h
OR
OMOM
OMe
OMe
OMe
OR
OMe
21
19 R = H
12
2
R = Bn
R = Na
11
c, d
20
3
R = Ser
R = Ser.HCl
i
h
e
MeO
MeO
MeO
MeO
MeO
MeO
MeO
MeO
j, k
j, k
O
O
OMe
OMe
OMOM
OMe
O
P
OR
OMe
NHR
i
NH
OH
OMe
OMe
OR
OMe
22 R = Boc
R = H
OMe
24
NH2
14
13
R = Bn
l
15 R = Na
l
=
25 24.HCl
23
Scheme 1. Synthesis of CA4 and cyclopropyl-containing derivatives. Reagents and
conditions: (a) TMS-diazomethane, n-BuLi, À78 °C, 90%; (b) MOMCl, DIPEA, 95%; (c)
triisopropylborate, n-BuLi, Pd(PPh3)4, 90 °C, 56%; (d) MeOH/HCl, 90%; (e) H2, Pd/
CaCO3, quinoline, 81%; (f) MeOH/HCl, 95%; (g) (BnO)2P(O)H, DIPEA, DMAP, 67%; (h)
(i) TMSBr, NaI, 48%; (ii) NaOMe, 70%; (i) CH2N2, Pd(OAc)2, 55%; (j) MeOH/HCl, 68%;
(k) (BnO)2P(O)H, DIPEA, DMAP, 67%; (l) (i) TMSBr, NaI; (ii) NaOMe, 32% (two steps).
Scheme 2. Preparation of N-containing combretastatin analogs. Reagents and
conditions: (a) Pd(PPh3)2Cl2, NEt3, CuI, 70%; (b) H2, Pd/CaCO3, quinoline, 83%; (c)
Fmoc-
90%; (g) H2, Pd/CaCO3, quinoline, 60%; (h) CH2N2, Pd(OAc)2, 58%; (i) MeOH/HCl, 85%;
(j) Fmoc- -Serin(Ac), DIC, HOBt, 76%; (k) NaOH, 92%, (l) HCl, 91%.
L-Serin(Ac), DIC, HOBt, 70%; (d) NaOH, 61%; (e) HCl, 91%; (f) Boc2O, K2CO3,
L

6950
R. Fürst et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6948–6951
Table 1
Calculated IC50 values for synthetic intermediates and final cyclopropyl derivatives of
combretastatin
Compound
Structural motif
Calculated IC50 value (lM)
Hela
MCF-7
9
10
17
18
1
Triple bond
Triple bond
Triple bond
Triple bond
26.81
1.54
>30
25.63
1.64
>30
>30
>30
Cis-double bond
Cis-double bond
Cis-double bond
Cs-double bond
Cis-double bond
Cis-double bond
Cis-double bond
Cis-double bond
Cyclopropyl ring
Cyclopropyl ring
Cyclopropyl ring
Cyclopropyl ring
Cyclopropyl ring
Cyclopropyl ring
Cyclopropyl ring
Cyclopropyl ring
0.00051
0.00043
0.0037
0.197
0.246
0.00053
0.00072
1.17
0.028
23.96
10.17
0.37
0.0024
0.0020
0.025
0.269
1.17
0.0035
0.0031
3.85
0.102
14.01
10.77
0.297
11.84
0.212
0.063
0.670
2
3
11
12
19
20
21
4
13
14
15
22
23
24
25
Figure 3a. Docking of cyclopropyl derivatives 4 into the colchicine binding site of
tubulin. Yellow: DAMA-colchicines, green: Compd 4.32
10.07
0.041
0.032
0.432
IC50 values in the nanomolar range. Highest level of activity was
observed in compounds which are currently employed in clinical
studies. Reference compounds 1 and 2 are equipotent as dephos-
phorylation takes place in the medium and 1 as active agent enters
the cells. If the free hydroxyl (or amino) functionality on ring C is
blocked by substituents lacking hydrogen bonding donors (11
and 21), the activity level drops. This result is in agreement with
findings by other groups.29
Substrates with the cyclopropyl unit as structural motif are sig-
nificantly less active than the corresponding stilbene derivatives
but still possess moderate to good cytotoxic properties. As pointed
out above, cyclopropyl derivatives were prepared and tested as
racemic mixtures and therefore the activity of the enantiopure
material is not known. However, since all racemic cyclopropyl
compounds are substantially less potent than the corresponding
stilbenes, even enantiopure substrates would express lower po-
tency than the natural product.
It is interesting that phosphate 15 is less active than 4 suggest-
ing that dephosphorylation proceeds at lower rate than in 2.
Again, the hydroxyl- or amino-functionality at ring C plays a
major role and the activity is much higher when hydrogen bonding
donors are available. Intermediates lacking acidic protons are
nearly inactive.
The serine moiety is cleaved rapidly in the stilbene- and cyclo-
propyl-series as 20 and 24 showed similar activities compared to
19 and 23.
We were surprised that the hydrochloric salts 3 and 25 were
significantly less active than the free amine. This observation is
poorly understood. Counter ions often play a major role in the po-
tency of biologically active compounds and protein binding is also
determined by ion concentration in the medium. A similar counter
ion depending activity was also observed by Pettit in the case of
phosphorylated combretastatins.30
Figure 3b. Docking of cyclopropyl derivatives 23 into the colchicine binding site of
tubulin. Yellow: DAMA-colchicines, green: Compd 23.32
alignment of the trimethoxy-phenyl rings but a different orienta-
tion of ring B pointing towards the ß-sheets with Lys352.
Despite the lower in vitro activity of cyclopropanes in compar-
ison to the natural product, cyclopropanes lack the ability to un-
dergo cis/trans isomerization and therefore represent highly
interesting lead compounds for in vivo studies. The spatial demand
of the cyclopropyl moiety is rather small and the incorporation of a
cyclopropane ring does not change the overall polarity of the sub-
strate. The main difference between natural stilbene derivatives
and the corresponding cyclopropanes emerges from different an-
gles between the aromatic rings. By preparing substrates with var-
ied ring size (cyclobutyl or cyclopentyl moieties), the angle of the
aromatic rings can be adjusted. Fine tuning of the spatial arrange-
ment of the aromatic rings by synthesis of such compounds and
subsequent evaluation of the biological activity is currently under
investigation in our laboratories and will be reported in due course.
Acknowledgments
Preliminary docking studies of compounds 4 and 23 into the
colchicine binding site of tubulin (pdb code 1sa0)31 further
strengthen the importance of H-bonds. The phenol 4 shows an
H-bond to Thr179 whereas the aniline 23 form an H-bond
Ser178. However, considering side chain rotamers compound 4
might additionally form an H-bond with Ser178 (Figs. 3a and 3b).
Docking studies also propose two different binding hypotheses
for combretastatin analogs. Phenol 4 aligns well with DAMA-col-
chicine, whereas aniline 23 adopts a conformation with good
The authors (R.F. and U.R.) thank the Fonds zur Förderung der
wissenschaftlichen Forschung (FWF) for financial support of this
work (Project FWF-R45-N19).
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(Chemical Computing Group). All amino acids in a distance of 4.5 Å to
colchicine were used for defining the receptor. Ligands were docked using the
Triangle Matcher as placement routine with the rotate bonds function
activated. Rescoring 1 was performed with London dG, GridMin was used for
refinement and Affinity dG was selected for rescoring 2. The top 10 ranked
poses for each ligand were analyzed.