Chondramide C: Synthesis, configurational assignment, and structure-activity relationship studies

Article abstract of DOI:10.1002/anie.200801156

(Chemical Equation Presented) Two solutions for one problem: Of the four isomers of chondramide C synthesized, the two shown in the scheme exhibit nearly the same conformation of the peptide segment and consequently unfold equal biological activities.

Full text of DOI:10.1002/anie.200801156

Communications
DOI: 10.1002/anie.200801156
Total Synthesis
Chondramide C: Synthesis, Configurational Assignment, and
Structure–Activity Relationship Studies**
Ulrike Eggert, Randi Diestel, Florenz Sasse, Rolf Jansen, Brigitte Kunze, and Markus Kalesse*
In 1995 the groups of Reichenbach and Höfle isolated the
family of chondramides.[1] Three years later it was reported
that chondramides induce the polymerization of G-actin.[2] In
contrast, other natural products isolated from myxobacteria
such as rhizopodin[3] or chivosazol[4] lead to destabilization
and reorganization of actin filaments. In both cases a collapse
of actin-dependent cellular processes is the result. Addition-
ally, cytokinesis at the end of cell division cannot be
completed and enlarged polynuclear cells develop.
The structure elucidation unraveled an 18-membered
macrocycle without providing the configuration of the six
stereocenters included. The family of chondramides is
characterized by different substituents at R1 and R2
(Scheme 1). Compared to the structurally related jaspamide
(jasplakinolide) an obvious similarity is the amino acid
sequence of the tripeptide. The apparent concurrence was
Scheme 1. Members of the chondramide–jaspamide family.
reinforced by the fact that the b-tyrosine subunit has the same
configuration as in jaspamide.[5] One significant difference
compared to jaspamide is the ring size (18- versus 19-
membered) and a different secondary hydroxy group in the
polyketide segment of chondramide. Parallel to our contri-
bution the group of Waldmann and Arndt completed the total
synthesis of chondramide C.[6]
First members of the jaspamide family were isolated by
Zabriskie et al.,[7] Crews et al.,[8] and Braekman et al.[9] and
attracted significant attention due to their ability to induce
the polymerization of the actin skeleton.[10] A thorough
investigation of their anti-proliferative activity revealed
activity against 36 solid tumors,[11] and consequently initiated
a variety of synthetic contributions.[12] Besides total synthesis,
research groups targeted simplified jaspamide analogues.
Riccio et al.[13] substituted the polyketide segment by an
amino acid linker. In a different approach, Maier et al.[14]
investigated the effect of allylic strain by impeding the
preferred conformation with the aid of arenes. Nevertheless,
all synthetic derivatives showed reduced biological activities.
Recently, extended structure–activity relationship (SAR)
studies became available when Zampella et al.[15] isolated
additional natural jaspamide derivatives from Jaspis splen-
dans. Nevertheless, a detailed investigation of the structural
significance of the polyketide segment of the jaspamides–
chondramides family still has not been reported.
Based on the structural similarities of the two classes we
started a synthesis program aimed at providing derivatives of
chondramide C that would help to determine the absolute
configuration of the chondramides and to provide first SAR
data. Furthermore, we proposed that the polyketide segment
determines the conformation of the tripeptide and is thus the
pivotal structural element for the biological activity.
[*] U. Eggert, Prof. Dr. M. Kalesse
Zentrum für Biomolekulare Wirkstoffe (BMWZ)
Leibniz Universität Hannover
The synthesis of the tripeptide segment is based on
procedures published in the context with the jaspamide
Schneiderberg 1B, 30167 Hannover (Germany)
Fax: (+49)511-762-3011
E-mail: Markus.Kalesse@oci.uni-hannover.de
and
Head of Department Medicinal Chemistry
Helmholtz Centre for Infection Research
Inhoffenstrasse 7, 38124 Braunschweig (Germany)
synthesis.[7,16,17] and commenced with compound
(Scheme 2).[16] The synthesis of tri-
peptide 14 has been described for
jaspamide by Kocienski et al. and
provides with exception of the bro-
mination step the basis for the tripep-
8
R. Diestel, Dr. F. Sasse, Dr. R. Jansen, Dr. B. Kunze
Helmholtz Centre for Infection Research
Inhoffenstrasse 7, 38124 Braunschweig (Germany)
tide synthesis of chondramide
C
(Scheme 3). The adjustments neces-
sary for our synthesis include the
synthesis of 10 according to Hirai
et al.[18] Here, in particular the use of
DMF as the solvent in the methyl-
[**] This work was supported bythe DFG (Ka 913/11-1). We thank
Bettina Hinkelmann and Birte Engelhardt for technical assistance
with the cellular assays.
Scheme 2. The b-tyro-
sine unit 8. TBS=tert-
butyldimethylsilyl.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200801156.
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Chemie
cyclization failed and to exclude the possibility that the
observed problems were due to unfavorable isomers we
optimized the cyclization protocol on the seco acid derived
from authentic chondramide C.
The macrolactonization provided unexpected challenges
because established protocols such as the Keck–Boden
method,[21] the Yamaguchi lactonization,[22] as well as the
Mukaiyama or Mitsunobu reaction[23] proved unsuccessful.
Other strategies such as transesterification using
[(nBu2ClSn)O] according to Giannis et al. and Otera
et al.[24] were similarly unsuccessful. Fortunately, the
Shiina[25] method using 2-methyl-6-nitrobenzoic anhydride
(MNBA) to activate the carboxylic acid provided acceptable
yields of all four stereoisomers (Scheme 4). Following the
route described above, the diastereomers differing in the
configurations of the polyketide segment were obtained and
comparison of their NMR spectra clearly identified isomer 20
to be identical with natural chrondramide C.
With these four isomers in hand we began to investigate
their biological activity. By comparing their anti-proliferative
effects with the ability to induce the polymerization of actin,
valuable information can be obtained not only in terms of
SAR studies, but also as to whether an additional cellular
Scheme 3. Synthesis of the tripeptide of chondramide C based on the
route devised byKocienski et al. [16] Boc=tert-butoxycarbonyl,
DCC=N,N’-dicyclohexylcarbodiimide, HOBT=1-hydroxy-1H-benzotri-
azole, LDA=lithium diisopropylamide, OTf=trifluoromethanesulfo-
nate, TBAF=tetrabutylammonium fluoride, TFA=trifluoroacetic acid.
ation step increases the yields to 84% over two steps. To
improve the non-reproducible yield in the deprotection step
(13!14) the sequence reported by Grieco et al.[12e] was
employed. In this two-step sequence, intermediate 13 was first
protected with TBS and then treated with two equivalents of
K2CO3 in THF/MeOH/H2O (2:1:1) at room temperature to
provide the liberated amine 14 in good yield.
The synthesis of the polyketide segment commences with
a syn-selective crotyl boration based on the protocol devised
by Brown et al.,[19,20] followed by protection by PMB and
ozonolysis. The resulting aldehyde was subjected to a Wittig
olefination and provided unsaturated ester 15. Reduction
with DIBAl-H and treatment with iodine, imidazole, and
PPh3 provided the iodide 16, which was employed in an Evans
alkylation to furnish 17. Liberation of the acid can be
achieved under standard conditions by using LiOH and
H2O2 (Scheme 4).
Tripeptide 14 was then coupled with acid 18 using DCC
and HOBT and the PMB group was removed by using BCl3.
Remarkably, attempts to perform this transformation with
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) failed and
provided oxidized tryptophan as the sole product. Liberation
of the corresponding seco acid was achieved by using LiOH in
EtOH/THF/H2O. Initial experiments to achieve the macro-
Scheme 4. Synthesis of the polyketide segment 18 and coupling with
the tripeptide. Bn=benzyl, DIBAl-H=diisobutylaluminum hydride,
DMAP=4-dimethylaminopyridine, DMS=dimethyl sulfide, MNBA=2-
methyl-6-nitrobenzoic anhydride, NaHMDS=sodium hexamethyldisila-
zanide, PMB=para-methoxybenzyl.
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Table 1: Antiproliferative activityof 20, 21, 22, and 23 on different mammalian cell lines, recorded as IC50
values in nm.[a]
diastereomers (see Supporting
Information) gave the following
approximate EC50 values: 2 (20), 3
(21), 5 (22), and 6 mm (23). The EC50
data are related to the maximum
value obtained by a sigmoidal fit of
the polymerization curve with 20
(40 mm). As shown in Figure 1 with
different concentrations of 20, the
relation between the concentration
Cell line
Origin
20
21
22
23
L-929
murine connective tissue fibroblasts
human epidermoid carcinoma
human kidneycarcinoma
human lung carcinoma
human ovaryadenocarcinoma
55ꢀ24
55ꢀ5
81ꢀ6
49ꢀ3
32ꢀ2
23ꢀ2
19ꢀ2
1670ꢀ390
620ꢀ280
500ꢀ130
510ꢀ195
320ꢀ60
2400ꢀ115
1200ꢀ45
920ꢀ270
930ꢀ60
A-431
A-498
A-549
SK-OV-3
24 ꢀ3
26ꢀ5
16 ꢀ5
1040ꢀ160
[a] Data are means ꢀ standard deviations of two independent IC50 value determinations.
target might be addressed that at least in part would account
for the observed biological activity.
To assess the anti-proliferative effects of chondramide C,
five different cell lines were investigated. As shown in Table 1
diastereomer 21 had a strong growth inhibitory activity on
cultured mammalian cells. The data obtained were nearly the
same as those observed for the natural chondramide C (20;
Scheme 5) isolated from Chondromyces crocatus. The diaste-
reomers 22 and 23 were clearly less efficacious in inhibiting
cell propagation; on the average their IC50 values were 20 and
36 times higher, respectively.
Figure 1. Induction of G-actin polymerization by chondramide. Pyrene-
actin in a nonpolymerizing buffer was incubated with chondramide C
diastereomers, and actin polymerization was monitored by measuring
increasing fluorescence. Each diastereomer was applied at a concen-
~
&
^
*
tration of 10 mm (depicted byblack symbols:
20, 21, 22, and
23). For chondramide C the induction efficacyat additional concen-
~
!
&
*
trations is shown (graysymbols:
methanol).
40 mm, 5 mm, and 1 mm;
and the percentage of polymerization induction is strongly
exponential. This has to be taken into account when compar-
ing the EC50 data.
With isomer 21 at a concentration of 160 nm, we clearly
observed effects on the actin cytoskeleton of kidney cancer
cells (Figure 2). Reorganization of the actin structures of the
cells during mitosis seemed to be impaired, and in the
interphase cells stronger stress fibers, spots, and flakes of F-
actin were formed. After 18 h the first cells with two nuclei
were observed. We could also demonstrate these phenotypes
with diastereomer 22 but only at much higher concentration
(8 mm).
The superimposition of the four diastereomers shows that
20 and 21 exhibit similar conformations in the peptidic part
even though two of the three configuration centers in the
polyketide segment display opposite configurations. On the
other hand the conformations of 22 and 23 deviate signifi-
cantly from those of 20 and 21 (Figure 3).
The unexpected similar biological profile of compounds
20 and 21 can be rationalized by a similar geometric constrain
imposed by the polyketide segment of both isomers. Scheme 6
depicts the conformation of the segments incorporated, and it
can be seen that the torsion angles and distances in 20 and 21
Scheme 5. Chondramide C diastereomers.
In a subsequent set of experiments the ability of chon-
dramide C to induce actin polymerization was determined.
Figure 1 shows that the natural compound 20 is more
efficacious than the other isomers, but also in these cell-free
experiments, diastereomer 21 was more active than 22 and 23.
Polymerization curves with different concentrations of the
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Scheme 6. Polyketide segments of compounds 20, 21, and 23.
pounds, the natural product 20 and isomer 21 exhibit nearly
the same biological activity. This can be explained through a
conformational analysis of the isomers, which clearly shows
that 20 and 21 exhibit nearly the same conformation of the
tripeptide. This analysis clearly supports the hypothesis that
the polyketide segment acts as a structural element that
performs a fine-tuning on the conformation of the amino
acids in the molecule.
Figure 2. Influence of 21 on the actin cytoskeleton of A-498 kidney
cancer cells after different incubation times. F-actin was labeled green,
nuclei and chromosomes blue. A, B: control cells with a mitotic cell in
the center; metaphase (A), and telophase (B). Cells that were
incubated with 21 (160 nm) showed abnormal metaphase cells (C,
after 2 h), and a strengthened contractile ring in the late telophase (E,
after 18 h). Spots of F-actin became visible especiallyat focal adhesion
points (D, after 4 h), stress fibers became stronger and flakes of actin
appeared (E and F, after 18 h).
Received: March 10, 2008
Revised: April 28, 2008
Published online: July 15, 2008
Keywords: chondramide · natural products ·
.
structure elucidation · structure–activityrelationships ·
total synthesis
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