Synthesis of a cytotoxic ansamycin hybrid

Article abstract of DOI:10.1021/ol5011278

The synthesis of a new ansamycin macrolactam derivative that contains an ansa chain based on ansamitocin and an aromatic core related to geldanamycin is reported. The selective introduction of the cyclic carbamoyl group at C7 and C9 relies on a biotransformation using a mutant strain of S. hygroscopicus, the geldanamycin producer. The ansamycin hybrid forms atropisomers that differ in their antiproliferative activity toward cancer cells.

Full text of DOI:10.1021/ol5011278

Letter
pubs.acs.org/OrgLett
Synthesis of a Cytotoxic Ansamycin Hybrid
Gerrit Jurjens and Andreas Kirschning*
̈
Institute of Organic Chemistry and Center of Biomolecuclar Drug Research (BMWZ), Leibniz Universitat Hannover, Schneiderberg
̈
1B, 30167 Hannover, Germany
S
* Supporting Information
ABSTRACT: The synthesis of a new ansamycin macrolactam derivative
that contains an ansa chain based on ansamitocin and an aromatic core
related to geldanamycin is reported. The selective introduction of the
cyclic carbamoyl group at C7 and C9 relies on a biotransformation using a
mutant strain of S. hygroscopicus, the geldanamycin producer. The
ansamycin hybrid forms atropisomers that differ in their antiproliferative
activity toward cancer cells.
he ansamycin antibiotics are a growing class of complex
macrolactam antibiotics that are produced by various
Although the maytansinoids and geldanamycin are structur-
ally closely related as far as ring-size and position of
functionalities are concerned, they target completely different
proteins. Therefore, we pursued the idea to combine structural
features recruited from both ansamycins antibiotics within one
molecule. Since the exact mode of binding to Hsp90 is known
for geldanamycin while unknown for the ansamitocins, we
targeted a macrolactam composed of an ansamitocin backbone
in order to maximize tubulin binding possibilities while the
aromatic core resembles that of geldanamycin. The target
hybrid ansamycins 6 and 7 would allow to participate in an H-
bonding network with Hsp90 similar to geldanamycin9 as
assumed from SAR studies published for both biological
targets.10,11 We decided to pursue a total synthesis approach as
we expected that late stage oxidation of the aromatic moiety of
AP3 2 would hardly be possible. Although supplementing the
AHBA(−) mutant strain of A. pretiosum with a derivative of the
biosynthetic starting building block 3,5-aminohydroxy benzoic
acid is a powerful strategy in accessing new ansamitocins, it
would fail here because substituents at C17 and C21 can not be
incorporated by mutasynthesis.12
T
actinomycetes, but in the special case of maytansine (4) they
can also occur in plants (Figure 1).1 The ansamycin antibiotics
Figure 1. Structures of ansamycin antibiotics of the maytansinoid
family 1−4, geldanamycin.
possess a cyclic structure that is characterized by an aliphatic
ansa chain that forms a bridge between two nonadjacent
positions at the aromatic or quinone core. Important examples
are the ansamitocins 1−3, maytansine (4),2,3 and geldanamycin
(5).4 The maytansinoids 1−4 exhibit strong antiproliferative
activities in the lower nanomolar to subnanomolar range. A
maytansin−antibody conjugate (trastuzumab emtansine; T-
DM1, trade name Kadcyla) was approved as a chemo-
therapeutic against HER2-positive breast cancers early last
year. While the maytansinoids bind to tubulin and inhibit its
polymerization, the cytotoxicity of geldanamycin 5 is linked
with the blocking of the ATP binding site of heat shock protein
Hsp90α.4
The concept of hybrid architectures based on natural
products has frequently been pursued in academia and the
pharmaceutical industry.5 The underlying idea is to develop a
molecule that contains two different biological activities, and
this is commonly achieved by linking two different small
molecule drugs or natural products. The most prominent
applications have been realized in conjugating antitumor drugs
with tumor-specific ligands such as folic acid6,7 or cyclic
peptides.8
Retrosynthetically, we planned to chemoselectively introduce
the cyclic carbamoyl group and the isobutyrate side chain at the
very end of the synthesis via a mutasynthetic operation using
our mutant strains of A. pretiosum or S. hygroscopicus. For
carrying out this mutasynthesis, proansamitocin derivative 8
became one key intermediate (Scheme 1).21 Macrolactam 8 is
f
further dissected into vinyl iodide 9 and the eastern ansachain
10 which could be merged by an intermolecular Heck
reaction13 followed by a Goldberg macrolactamization.14
Importantly, Panek and co-workers demonstrated the feasibility
of the Goldberg macrolactamization with electron rich aromatic
systems in their total synthesis of geldanamycin.15 We planned
to access pentasubstituted arene 9 from compound 11. We also
planned to employ a vinylogous aldol reaction as key step for
the synthesis of the ansa chain 10.16 For achieving this highly
Received: April 18, 2014
© XXXX American Chemical Society
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Scheme 1. Retrosynthetic Plan
Scheme 3. Synthesis of the C7−C12 Fragment22
moderate yield when aldehyde 21 was coupled with vinylogous
silyl enol ether 22 (Scheme 4). We faced difficulties with
Scheme 4. Two Synthetic Approaches toward the C3−C12
Fragment
convergent step, facile access to enol 12 and aldehyde 13 was
necessary. Aldehyde 13 was chosen as a key building block
because alternative synthetic routes could be envisaged from 13
if the vinylogous aldol reaction would turn out to be
problematic.
The synthesis of the western fragment commenced from
hydroquinone 11 (Scheme 2). A sequence of protection,
Scheme 2. Synthesis of the Aromatic Fragment 9
formylation, and bromination yielded the pentasubstituted
aromatic core 14.15 Reduction followed by Appel-type reaction
then provided compound 15. Palladium-catalyzed coupling of
15 with an alkynyl zinc reagent,17 followed by mild desilylation,
provided compound 16. Finally, zirconium-mediated carboalu-
mination18 followed by iodination provided vinyl iodide 9.
The synthesis of aldehyde 21 started with the Michael
addition of p-methoxybenzyl acohol 17 to acroleine 18
(Scheme 3).19 The resulting aldehyde was subjected to a
Brown allylation20 using the Z-configured allyl borane 19,
which furnished alcohol 20 with high stereocontrol. Mosher
ester analysis confirmed the absolute configuration of the
carbinol moiety.21
reproducibility on large scale, and substantial optimization (e.g.,
Lewis acids, choice of silyl group) did not improve the results.
The configuration of the newly formed carbinol moiety was
confirmed by Mosher ester analysis and through chemical
correlation using material obtained through the independent
route (Scheme 4). This second route involved a syn Evans aldol
reaction followed by protection of the secondary alcohol and
reductive cleavage of the auxiliary to provide alcohol 25. A
sequence of oxidation, Wittig olefination, and reduction then
furnished allylic alcohol 26.
Concerning the vinylogous aldol reaction, we found that
there the choice of Lewis acid is delicate. Strong Lewis acids
such as TiCl4 enforce degradation, while milder Lewis acids did
not lead to full conversion and reactants 21 and 22 were
recovered in high yield. The synthesis of the polyketide
For assembling the carbon backbone of C3−C1022 aldehyde
21 was subjected to a vinylogous aldol protocol16 based on
Kobayashi’s work.23 Thus, formation of amide 23 proceeded in
B
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ansachain was finalized by oxidation of the allylic alcohol 26 to
the aldehyde 27 followed by an aldol reaction using a BSA-
derived lithium enolate.24 The diasterofacial selectivity of this
substrate-controlled process was good at low temperatures and
gave the desired 3S-configured diastereoisomer. The preference
for si-face attack can be rationalized from the preferred
conformation of aldehyde 27 (Figure 2), which minimizes
tailoring modifications on macrolactam 31 despite the fact
that the ansachain in 31 closely resembles proansamitocin.12f
Finally, introduction of the ester side chain provided ansamycin
derivative 7 (Scheme 5), but all attempts to remove the
isopropyl groups and generate hydroquinone 6 failed, an
observation that Bach et al. also noted recently.28,29
Scheme 5. Chemoenzymatic Endgame of Ansamycin
Synthesis (7a and 7b Refer to Atropisomers)
Figure 2. Lowest energy conformation of 27.25
the allylic strain of the methyl groups in C4 and C6.22 TBS-
protection and radical-initiated removal of the benzyl
protecting group yielded the C1−C11 fragment 10 (Scheme
4).
The diasterofacial selectivity of this substrate-controlled
process was good at low temperatures and gave the desired
3S-configured diastereoisomer. The preference for si-face attack
can be rationalized from the preferred conformation of
aldehyde 27 (Figure 3), which minimizes the allylic strain of
Compounds 32 and 7 demonstrated atropisomerism for
which the two isopropoxy groups can be made responsible.
One of the two atropisomer of 32 and 37 was conformationally
stable and could be separated via HLPC and spectroscopically
characterized. In contrast, the other atropisomers showed broad
1
lines in the H NMR spectra likely due to the presence of
several conformers.
Comparison of the NMR data suggest that the stable
atropisomers exhibit a similar conformation to ansamitocin P3
(2) in solution.
Figure 3. Antiproliferative activities of 7 and of ansamitocin derivatives
structurally related to derivative 7.
the methyl groups in C4 and C6.25 TBS-protection and radical-
initiated removal of the benzyl protecting group yielded C1−
C11 fragment 10 (Scheme 4).
Heck coupling under Jeffrey conditions yielded seco
carboxamide 28, which was cyclized under Goldberg
conditions. Surprisingly, the resulting macrolactam 29 was
present as two atropisomers. These could be separated
chromatographically, and the thermodynamically less favored
could be converted into the other one when being heated in
DMF at 145 °C.
We had to test a series of oxidants (e.g., DMP, Swern, TPAP,
Oppenauer) to establish the keto functionality at C9 before
PDC and molecular sieves (3 Å) were found to be best suited
to deliver compound 30.26 During these trials, we observed
decomposition or oxidation of the electron-rich aromatic
moiety. Ketone 30 was desilylated using PPTS, which provided
macrolactam 31. The removal of the bulky silyl groups
provided rotational flexibility in the macrocycle and no
conformationally stable atropisomers could be detected any-
more. The carbamoyl group was selectively introduced by a
mutasynthetic biotransformation27 using an aminohydroxyben-
zoic acid blocked mutant of the geldanamycin producer S.
hygroscopicus12f which provided ansamycin derivative 32.
Surprisingly, the corresponding AHBA(−) mutant of the
ansamitocin producer A. pretiosum did not perform any
Biological evaluation conducted with the conformationally
stable atropisomers of 7 and 32 revealed no antiproliferative
activity toward mouse fibroblasts L929 and selected cancer
lines, which contrasts other very potent ansamitocin derivatives
that also lack the N-methyl group and the oxirane ring (for
selected data, see Figure 3). To our surprise, the minor
atropisomers of 7 and 32 exhibit a pronounced acitivity against
cervix carcinoma cells KB-3−1 (2.2 and 5.0 μg mL−1,
respectively) as well as against ovary adenocarcinoma cells
SKOV-3 (4.5 and 7.2 μg mL−1, respectively). Since these
isomers conformtionally do not resemble ansamitocin it is likely
that the antiproliferative activity is based on a completely
different biological mechanism, particularly as they also showed
no inhibitory activity against Hsp90α in a competition assay
with fluorescent-labeled ATP.25
In conclusion, we accomplished the synthesis of the first
ansamitocin hybrid derivative 7 that resembles the aromatic
substitution pattern of geldanamycin. Key steps are a highly
selective mutabiosynthetic step, a Goldman cyclization, and a
Heck coupling of the two main fragments. The biological
evaluation unexpectedly revealed that one of two atropisomers
irrespective whether acylated at C3 or not shows antiprolifer-
ative activity against selected cancer cell lines but does not
target Hsp90α. As all tubulin-addressing ansamitocins require
C
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derivative addressing a new biological target. It needs to be
noted that the conformational flexibility of these atropisomers
are important for being able to adapt to a biological target
including a new one. Our current studies are directed toward
elucidating the biological target of macrolactam 7 and
analogues.
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This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*E-mail: andreas.kirschning@oci.uni-hannover.de.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The work was funded by the Deutsche Forschungsgemeinschaft
(Grant No. Ki 379/16-1). We thank F. Sasse (Helmholtz
Center of Infectious Research (HZI), Braunschweig, Germany)
for conducting antiproliferation tests and C. Zeilinger (Institute
of Biophysics, Leibniz Universat Hannover, Germany) for
̈
carrying out Hsp90α inhibition tests.
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