Catalytic Asymmetric Total Synthesis and Stereochemical Revision of Leucinostatin A: A Modulator of Tumor–Stroma Interaction

Article abstract of DOI:10.1002/chem.201703239

Total synthesis of leucinostatin A, a modulator of tumor-stroma interactions, using asymmetric catalyses, a nitroaldol reaction, thioamide-aldol reaction, Strecker-type reaction, and alcoholysis of 3-methylglutaric anhydride, is described. We demonstrated the applicability of the established catalytic asymmetric processes to the synthesis of molecules with a complex structure. Careful analysis of the NMR data, HPLC profiles, and biological activity revealed that the correct structure of leucinostatin A is the epimeric form of the reported structure; the secondary alcohol within the AHMOD residue has an R configuration.

Full text of DOI:10.1002/chem.201703239

A Journal of
Accepted Article
Title: Catalytic Asymmetric Total Synthesis and Stereochemical
Revision of Leucinostatin A, a Modulator of Tumor-Stroma
Interaction
Authors: Masakatsu Shibasaki, Hikaru Abe, Hitoshi Ouchi, Chiharu
Sakashita, Manabu Kawada, and Takumi Watanabe
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To be cited as: Chem. Eur. J. 10.1002/chem.201703239
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Catalytic Asymmetric Total Synthesis and Stereochemical
Revision of Leucinostatin A, a Modulator of Tumor-Stroma
Interaction
Hikaru Abe, Hitoshi Ouchi, Chiharu Sakashita, Manabu Kawada, Takumi Watanabe,* Masakatsu
Shibasaki*
Abstract: Total synthesis of leucinostatin A, a modulator of tumor-
stroma interactions, using an asymmetric catalysis, a nitroaldol
reaction, thioamide-aldol reaction, Strecker-type reaction, and
alcoholysis of 3-methylglutaric anhydride, is described. We
demonstrated the applicability of the established catalytic asymmetric
process to the synthesis of molecules with a complex structure.
Careful analysis of the NMR data, HPLC profiles, and biological
activity revealed that the correct structure of leucinostatin A is the
epimeric form of the reported structure; the secondary alcohol within
the AHMOD residue has an R-configuration.
to inhibit the growth of DU-145 prostate cancer cells more potently
under coculture with prostate stromal cells (PrSC) than under
monoculture conditions. Intriguingly, leucinostatin
A exerts
antitumor activity in vivo in mouse only when coinoculated with
DU-145 cells and PrSC.
Leucinostatin A is comprised of 4-methyl-2-hexenoic acid (ΔMHA),
4-methylproline
(MePro),
2-amino-6-hydroxy-4-methyl-8-
oxodecanoic acid (AHMOD), threo--hydroxyleucine (HyLeu),
three 2-aminoisobutyric acid (Aib), two leucines (Leu), -alanine
(-Ala), and N1,N1-dimethylpropane-1,2-diamine (DMPD).[6],[7]
Cerrini and co-workers reported the crystal structure of
leucinostatin A to assign the S-configuration of the secondary
alcohol within the AHMOD residue,[8] which could not be unveiled
in the previous degradation study. Recently, Brimble and co-
workers reported the total synthesis of peptibol antibiotics
containing the AHMOD residue such as culicinin D,[9] and
trichoderin A,[10] however, no precedent for leucinostatin A has
been reported to date. Herein we disclose the first catalytic
asymmetric total synthesis of leucinostatin A, which exemplifies
the general applicability of four catalytic asymmetric processes
consisting of three C-C bond-forming reactions as well as one C-
O bond formation developed in this laboratory in order to access
complicated chemical entities.
Figure 1. Reported structure of leucinostatin A
Tumor cells never exist in isolation, and are surrounded by normal
cells such as fibroblasts (stromal cells).[1] Tumor cell proliferation
is regulated by the stromal cells through secreted factors and
adhesion:[2] an intercellular communication of this type is termed
tumor-stroma interactions. The signals involved in tumor cell
growth emitted from stromal cells are a promising target in the
current anticancer lead generation process as normal cells are
expected to be less prone to mutation of proteinous factors and
acquisition of resistance due to the stable nature of the genes. We
have investigated modulators of tumor-stroma interactions among
natural products, especially those produced by microorganisms
and fungi. In our assay system, the hit compounds should
selectively suppress the proliferation of tumor cells cultured in the
presence of the corresponding stromal cells as opposed to their
absence.[3] During screening, leucinostatin A (Figure 1) produced
by fungi,[4] which had been originally reported as an effective
antibacterial and cytotoxic peptidyl natural product,[5] was found
Scheme 1 illustrates our synthetic strategy focusing on the
unnatural amino acid components, HyLeu and AHMOD. A
nitroaldol process could set up the carbon framework of HyLeu
from inexpensive substrates 4 and 5. It was proposed that the
AHMOD unit could be constructed with a three-catalyst protocol:
the the 4-methyl substituent could be furnished by asymmetric
methanolysis of 3-methylglutaric anhydride 13 to provide the
corresponding half ester 12, and S-configuration of the -carbon
atom should be delivered by Strecker-type reaction from 11 to 10,
followed by a thioamide-aldol reaction of 8 and 9 to install the (S)-
hydroxy group at 6-position in the adduct 7. The two aldol-type
methodologies exemplify direct catalytic asymmetric reactions; in
the C-C bond-forming process, only proton-transfer from the
prenucleophile to the hydroxy group of the product occurs and no
pre-activation of the substrate by derivatization is required, which
fulfils perfect atom economy.
[*]
H. Abe, Dr. H. Ouchi, C. Sakashita, Dr. M. Kawada, Dr. T.
Watanabe, Prof. Dr. M. Shibasaki
Institute of Microbial Chemistry (BIKAKEN), Tokyo
Address 3-14-23 Kamiosaki Shinagawa-ku, Tokyo, 141-0021
(Japan)
E-mail: mshibasa@bikaken.or.jp, twatanabe@bikaken.or.jp
Supporting information for this article is given via a link at the end of
the document.
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Scheme 1. Strategy toward catalytic asymmetric total synthesis of leucinostatin
A.
Scheme 2. Synthesis of HyLeu segment.
The next synthetic task was preparation of the AHMOD segment:
several synthetic studies related to AHMOD distinct from
leucinostatin A have been reported.[9,12] Our synthesis was
initiated (Scheme 3) from the central part of AHMOD; the 4-methyl
group was furnished with good enantiocontrol in the first step by
catalytic asymmetric methanolysis of 3-methylglutaric anhydride
13, which led to the half ester 12 as reported previously (93%
ee).[13] The carboxyl functionality of 12 was readily converted to a
formyl group (17) according to the reported protocol,[14] which was
followed by Wittig-type chemistry to provide trisubstituted olefin
18. The remaining ester moiety was subsequently transformed
into imine to give the substrate (11) of a Strecker-type reaction.
As detailed in Scheme 2, the present total synthesis commenced
with preparation of HyLeu. Although this segment is commercially
available, we envisioned that a catalytic asymmetric syn-selective
nitroaldol reaction could be used to construct the requisite carbon
skeleton with high enantioselectivity. Indeed, LLB* (a modified
LLB complex as shown in Scheme 2) is a particularly effective
catalyst to facilitate highly enantioselective nitroaldol reactions
using 2-nitroethanol 4 as a substrate.[11] In the original report, no
-branched aliphatic aldehyde was examined. Therefore, we
optimized the reaction conditions for isobutyraldehyde 5 (Table
S1). It was found that 9 mol % of LLB* catalyst with 3:1 ratio of 4
and 5 in THF at −40 °C for 140 h afforded a good yield (86%) and
satisfactory diastereo- (12:1) and enantioselectivity (96% ee) of
the corresponding nitroaldol adduct 3. At this stage, the
enantiopurity of 3 could be enriched to > 99% ee by recrystallization.
Progression to HyLeu derivative 2, which may be used in Fmoc-
based peptide synthesis, was performed without trouble. The nitro
group of 3 was reduced to a primary amine, followed by Fmoc-
protection to give 14. The secondary alcohol was then selectively
masked as a TBS ether to give 16 via bissilyl ether 15. Finally, a
series of oxidation protocols (Dess-Martin, and Pinnick
oxidations) completed the catalytic asymmetric synthesis of 2.
Scheme 3. Synthesis of the substrate of the thioamide-aldol reaction.
After optimization (Table S2) of the Strecker-type reaction, the
highest diastereoselectivity up to 5.5:1 (74% yield) was obtained
when 9 mol % of catalyst 19, 3 mol% of H2O, 0.2 equiv of phenol,
and 2 equiv of TMSCN were employed. Subsequently (Scheme
4), the cyano group of 10 was directly converted into methyl ester
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20 by treatment with 4 M HCl in a 1,4-dioxane-MeOH system
under gentle warming. The amino functionality was then bis-
protected by Boc groups (21) to avoid participation of the nitrogen
in an undesired cyclization with the aldehyde after the subsequent
transformation. Finally, ozonolysis afforded 9 in 80% yield.
transformation proceeded by Me3SnOH[16] giving 6 with no trace
of undesired byproducts.
With the requisite monomer units in hand, hexapeptide 25 was
synthesized on solid support (Supporting Information), and
progression to leucinostatin A was performed with HATU-
mediated[17] peptide coupling in liquid phase (HATU: 1-
[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxid hexafluorophosphate). Firstly, condensation
with DMPD (26) afforded 27 (Scheme 5). After cleaving the Fmoc
group, HyLeu-segment 2 was attached giving 28, then sequential
deprotection and coupling with AHMOD-segment 6 provided 29
(56%). Lewis acidic Boc-deprotection mediated by BF3·OEt was
found the most preferable[18] in order to avert undesired -
elimination of the siloxy moiety on AHMOD which was observed
when using standard TFA conditions. The final coupling reaction
with an acylated MePro, 30, accompanied deprotection to
complete the synthesis of leucinostatin A (1) in 43% yield. The
NMR data of the synthetic sample was barely distinguishable from
that of the natural compound (Figure S2). The antiproliferative
activity against DU-145 cells cocultured with PrSC (Table 1) of the
synthesized leucinostatin A, however, was 2-fold less potent than
the natural sample (IC50; natural: 0.028 ± 0.004 g mL-1,
synthetic: 0.063 ± 0.015 g mL-1), and the HPLC retention time
differed slightly (Figure S3). In the synthesis of culicinin D and
trichodermin A, Brimble concluded that the 6-position of AHMOD
bearing a hydroxy group has an R-configuration,[9,10] which is
opposite to the reported configuration. In each case, the
configuration was determined by comparing similarity of NMR
data of the R- and S-isomers to the reported values: in fact, exact
match was not observed for both the isomers. Although
complicated by the close similarity of the NMR data of the
synthetic and natural samples in our case and general reliability
of the configuration determined by crystallography,[8] we
synthesized the epimer of the reported leucinostatin A (epi-1 in
Scheme 5) for the analysis.
Scheme 4. Synthesis of AHMOD segments.
For installation of the -hydroxyketone moiety of AHMOD, a
catalytic asymmetric direct thioamide-aldol reaction condition was
effective (Scheme 4).[15] In this reaction, CuI complexed with a
chiral bidentate phosphine ligand, Ph-BPE, works as the soft
Lewis acid to activate thioamide 8 to promote deprotonation at the
-carbon, even in the presence of a more acidic proton adjacent
to the formyl group of 9. Interestingly, facial selectivity in this case
was opposite to the prediction based on the original report, which
led us to use (R,R)-Ph-BPE to set the S-configuration for the 6-
hydroxy functionality. After optimization (Table S3), the use of 5
mol% catalyst with chromanol as additive (22) in DMF at −60 °C
was revealed to give the best result. In the previous study, the
thioamide functionality could only be converted to methylketone
or -ketoester via the addition of lithium reagents to the
methylthioiminium intermediate generated by the treatment of
MeOTf.[15] In the present synthesis, EtMgBr was successfully
used for the first time, after protection of the hydroxy group as a
TBS ether (23). Although addition of Grignard reagent took place
without issue, subsequent hydrolysis by the original procedure
was slow: acid-treatment (1 M HCl/THF or NH4Cl) was required
instead of wet silica gel (original) to afford 24 in reasonable yield
(43%). The conversion of methyl ester to carboxylic acid under
conventional methods caused predominatly -elimination of the
siloxy functionality and epimerization at the -carbon. The desired
Table 1. Antiproliferative activity (IC50 (g mL-1)) of nominal leucinostatin A
and epimer toward DU-145 cells cocultured with PrSC cells.
Compound
Cocultured
Monocultured
~1
leucinostarin
A (natural)
0.028 ± 0.004
1
~1
~1
0.063 ± 0.015*
0.029 ± 0.001
epi-1
The values are means ± s.d. of three independent experiments with similar
results. *P < 0.01 versus the values of leucinostatin A (natural) (Student’s t-
test).
Performing the diastereoselective thioamide-aldol reaction of 8
and 9 using (R,R)-Ph-BPE (Scheme 4) gave aldol adduct epi-7
(86%) with
a
slightly compromised but still excellent
diastereoselectivity of 11:1. Subsequent transformations were
executed in the same manner as for 6 to afford epi-6, which was
coupled with the Fmoc-removed form of heptapeptide 28 to give
epi-29 (46%). The final coupling with 30 proceeded with
concomitant deprotection to provide an epimeric form of the
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reported structure of leucinostatin A, epi-1 (32%). The
physicochemical properties of epi-1 exactly matched those of
natural leucinostatin A including NMR data (Figure S4, also
almost very similar to that of 1 in Figure S2!). The retention times
of epi-1 and naturally occurring leucinostatin A based on HPLC
analysis were completely identical (Figure S5). Moreover, these
two samples exhibited the same growth inhibitory activity toward
DU-145 cells in the presence of PrSC (IC50; epi-1: 0.029 ± 0.001
g mL-1, Table 1). Together, this evidence led us to conclude that
the correct absolute configuration of the secondary alcohol of
AHMOD should be R, which is epimeric to the originally proposed
configuration.
In summary, the first total synthesis of leucinostatin A, a
modulator of tumor-stroma interactions, was achieved by applying
four catalytic asymmetric reactions developed in this laboratory,
which exemplify the adaptability of our processes with fine-tuning
to the synthesis of molecules with a complex structure. Careful
analysis of the NMR data, HPLC profiles, and biological activity
unequivocally indicated that the correct structure of leucinostatin
A is the epimer of the reported structure, with R-configuration at
the secondary alcohol portion of the AHMOD residue. Further
SAR studies to generate anticancer leads and probe compounds
to explore molecular targets upon intervening with tumor-stroma
interactions are in high demand and are currently underway.
Scheme 5. Completion of the total synthesis of leucinostatin A and stereochemical revision.
Keywords: Asymmetric catalysis • Total synthesis •
Acknowledgements
Configuration determination • Natural products• Tumor−stroma
interaction
M.S. is grateful to the JST (ACT-C), H. A. thanks the Uehara
Memorial Foundation, and T. W. thanks the KAKENHI (17K08384,
JSPS), the NOVARTIS Foundation (Japan) for the Promotion of
Science, The Sumitomo Foundation, Astellas Foundation for
Research on Metabolic Disorders, and Suzuken Memorial
Foundation for financial support. The authors also thank Dr. Isao
Momose (BIKAKEN) for providing natural leucinostatin A, Dr.
Ryuichi Sawa, Ms. Yumiko Kubota, Dr. Kiyoko Iijima, and Ms.
Yuko Takahashi (BIKAKEN) for the spectroscopic analysis, and
Ms. Maya Umekita (BIKAKEN) for HPLC analysis.
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Entry for the Table of Contents
COMMUNICATION
Total synthesis of leucinostatin A, a
modulator of tumor-stroma interaction
was achieved by applying four
H. Abe, H. Ouchi, C. Sakashita, M.
Kawada, T. Watanabe*, M. Shibasaki*
Page No. – Page No.
catalytic asymmetric reactions.
Analysis of NMR data, HPLC profiles,
and biological activity revealed that
the correct structure of leucinostatin A
is the epimer of the reported structure.
Catalytic Asymmetric Total Synthesis
and Stereochemical Revision of
Leucinostatin A, a Modulator of
Tumor-Stroma Interaction
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